Self-limiting viral vectors encoding nucleases

ABSTRACT

Disclosed herein are viral vectors for use in recombinant molecular biology techniques. In particular, the present disclosure relates to self-limiting viral vectors comprising genes encoding site-specific endonucleases as well as recognition sequences for site-specific endonucleases such that expression of the endonuclease in a cell cleaves the viral vector and limits its persistence time. In some embodiments, the viral vectors disclosed herein also carry directives to delete, insert, or change a target sequence.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/182,186, entitled “Self-Limiting Viral Vectors Encoding Nucleases,”filed Jun. 19, 2015, the disclosure of which is hereby incorporated byreference in its entirety.

FIELD OF THE INVENTION

The invention relates to the field of molecular biology and recombinantnucleic acid technology. In particular, the invention relates toself-limiting viral vectors comprising genes encoding site-specificendonucleases as well as recognition sequences for site-specificendonucleases such that expression of the endonuclease in a cell cleavesthe viral vector and limits its persistence time. Such viral vectors mayalso carry directives to delete, insert, or change a target sequence.Moreover the self-limiting viral vectors may be engineered to addresskinetic balancing (i.e., ensuring adequate expression of theendonuclease before that endonuclease finds its recognition sequencewithin the viral vector).

BACKGROUND OF THE INVENTION

AAV. Adeno-associated virus (AAV) is a small virus, which infects humansand several other primate species. AAV is not known to cause disease,and generally causes only a mild immune response. The virus infects bothdividing and quiescent cells and can be engineered to persist in anextrachromosomal state without integrating into the genome of the hostcell (Russel DW, Deyle DR (2010) Current Opinion in Molecular Therapy.11: 442-447; Grieger JC, Samulski RJ (2005) Advances in BiochemicalEngineering/Biotechnology 99: 119-45P). These features make AAV a veryattractive candidate for creating viral vectors for gene therapy. Recenthuman clinical trials using AAV for gene therapy in the retina haveshown promise (Maguire AM, et al. (2008) New England Journal of Medicine358: 2240-8). Moreover, AAV presents a well-known system with anestablished safety record with the completion of over sixty clinicaltrials. (Mitchell AM, Nicolson SC, Warischalk JK, and Samulski RJ (2010)Curr Gene Ther. 10(5): 319-40).

Wild-type AAV has the ability to stably integrate into the host cellgenome at a specific site (designated AAVS1) in the human chromosome 19.This feature makes it somewhat more predictable than other viral vectorssuch as retroviruses, which present the threat of random insertion andof mutagenesis. Gene therapy vectors based on AAV, however, generallyeliminate this integrative capacity by removal of the rep and cap genesfrom the DNA of the vector. In their place, a gene of interest can becloned under the control of a promoter between the viral invertedterminal repeats (ITRs) that aid in concatamer formation in the nucleusafter the single-stranded vector DNA is converted by host cell DNApolymerase complexes into double-stranded DNA. AAV-based gene therapyvectors form episomal concatamers in the host cell nucleus. Innon-dividing cells, these concatemers remain intact for the life of thehost cell. In dividing cells, AAV DNA is lost through cell division,since the episomal DNA is not replicated along with the host cell DNA.Random integration of AAV DNA into the host genome is detectable butoccurs at very low frequency.

AAV presents disadvantages as well. The cloning capacity of the vectoris relatively limited and most therapeutic genes require the completereplacement of the virus’s 4.7 kilobase genome. Large genes are,therefore, not suitable for use in a standard AAV vector. Options arecurrently being explored to overcome the limited coding capacity. TheAAV ITRs of two genomes can anneal to form head to tail concatamers,almost doubling the capacity of the vector. Insertion of splice sitesallows for the removal of the ITRs from the transcript, alleviatingconcatamer formation.

Because of AAV’s specialized gene therapy advantages, researchers havecreated an altered version of AAV termed self-complementaryadeno-associated virus (scAAV). Whereas AAV packages a single strand ofDNA, and must wait for its second strand to be synthesized, scAAVpackages two shorter strands that are complementary to each other. Byavoiding second-strand synthesis, scAAV can express more quickly, butalthough as a caveat, scAAV can only encode half of the already limitedcapacity of AAV (McCarty DM, Monahan PE, Samulski RJ (2001) Gene Therapy8: 1248-54)

The AAV genome is built of single-stranded deoxyribonucleic acid(ssDNA), either positive- or negative-sensed, which is about 4.7kilobase long. The genome comprises inverted terminal repeats (ITRs) atboth ends of the DNA strand, and two open reading frames (ORFs): rep andcap. The former is composed of four overlapping genes encoding Repproteins required for the AAV life cycle, and the latter containsoverlapping nucleotide sequences of capsid proteins: VP1, VP2 and VP3,which interact together to form a capsid of an icosahedral symmetry(Carter, BJ (2000) In DD Lassic & N Smyth Templeton. Gene Therapy:Therapeutic Mechanisms and Strategies. New York City: Marcel Dekker,Inc. pp. 41-59).

The Inverted Terminal Repeat (ITR) sequences comprise approximately 145bases each. The first 125 nucleotides of the ITR sequence arepalindromic, folding in on itself to create a T-shaped hairpin structure(Daya, Shyam (2008) Clin. Microbiol. Rev. 21(4) 583-593). The other 20bases of the ITR remain unpaired and are known as the D sequence. Theorigin of replication is the ITR and serves as a primer forsecond-strand synthesis.

With regard to gene therapy, ITRs seem to be the only sequences requiredin cis next to the therapeutic gene: structural (cap) and packaging(rep) proteins can be delivered in trans. With this assumption, manymethods have been established for the efficient production ofrecombinant AAV (rAAV) vectors containing a reporter, or therapeuticgene. However, it was also published that the ITRs are not the onlyelements required in cis for effective replication and encapsidation.Some research groups have identified a sequence designated cis-actingRep-dependent element (CARE) inside the coding sequence of the rep gene.CARE was shown to augment amplification, when present in cis (Nony P,Tessier J, Chadeuf G, et al. (2001) Journal of Virology 75: 9991-4).

On the “left side” of the genome, the rep genes are transcribed from twopromoters, p5 and p19, from which two overlapping messenger ribonucleicacids (mRNAs) of different length can be produced. Each of thesecontains an intron, which may or may not be spliced out. Given thesepossibilities generated by such a system, four various mRNAs, andconsequently, four various Rep proteins with overlapping sequence can besynthesized. Their names depict their sizes in kilodaltons (kDa): Rep78,Rep68, Rep52 and Rep40 (Kyöstiö SR, et al. (1994) Journal of Virology68: 2947-57). Rep78 and 68 can specifically bind the hairpin formed bythe ITR in the self-priming act and cleave at a specific region,designated terminal resolution site, within the hairpin. They were alsoshown to be necessary for the AAVS 1-specific integration of the AAVgenome. All four Rep proteins bind ATP and possess helicase activity. Asdemonstrated, Rep proteins upregulate the transcription from the p40promoter (mentioned below), but downregulate both p5 and p19 promoters.

The “right side” of a positive-sensed AAV genome encodes overlappingsequences of three capsid proteins, VP1, VP2 and VP3, which start fromone promoter, designated p40. The molecular weights of these proteinsare 87, 72 and 62 kiloDaltons, respectively. All three are translatedfrom one mRNA, the unspliced transcript producing VP1. After this mRNAis synthesized, it can be spliced in two different manners: either alonger or shorter intron can be excised resulting in the formation oftwo pools of mRNAs: a 2.3 kb- and a 2.6 kb-long mRNA pool. Generally,especially in the presence of adenovirus, the longer intron ispreferred, so the 2.3-kb-long mRNA represents the so-called “majorsplice.” In this form, the first AUG codon that initiates synthesis ofVP1 protein is cut out, resulting in a reduced overall level of VP1protein synthesis. The first AUG codon that remains in the major spliceis the initiation codon for VP3 protein. However, upstream of that codonin the same open reading frame lies an ACG sequence (encoding threonine,and serving as the initiation codon for VP2) surrounded by an optimalKozak context. This contributes to a low level of synthesis of VP2protein, which is actually VP3 protein with additional N terminalresidues, as is VP1. Since the bigger intron is preferred to be splicedout, and since in the major splice the ACG codon is a much weakertranslation initiation signal, the ratio at which the AAV structuralproteins are synthesized in vivo is about 1:1:20, which is the same asin the mature virus particle (Rabinowitz JE, Samulski RJ (2000) Virology278: 301-8).

The unique fragment at the N terminus of VP1 protein possessesphospholipase A2 (PLA2) activity, likely required for releasing the AAVparticles from late endosomes. VP2 and VP3 are crucial for correctvirion assembly (Muralidhar S, Becerra SP, Rose JA (1994), Journal ofVirology 68: 170-6). More recently, however, Warrington et al. haveshown VP2 to be not only unnecessary for the complete virus particleformation and an efficient infectivity, but that VP2 can tolerate largeinsertions in its N terminus (Warrington KH, et al. (2004), Journal ofVirology 78: 6595-609). In contrast. VP1 shows no such tolerance,probably because of the presence of the PLA2 domain (Id.). The AAVcapsid is composed of 60 capsid protein subunits, VP1, VP2, and VP3,that are arranged in an icosahedral symmetry in a ratio of 1:1:10, withan estimated size of 3.9 MegaDaltons. The crystal structure of the VP3protein was determined by Xie, Bue, et al. (Xie Q, Bu W, Bhatia S, etal. (2002) Proceedings of the National Academy of Sciences of the UnitedStates of America 99: 10405-10)

Currently, 12 AAV serotypes and nearly 100 variants have been identifiedin human and nonhuman primate populations. (Gao G, Zhong L, Danos O(2011) Methods Mol. Biol. 807:93-118). Serotypes can infect cells frommultiple diverse tissue types. Tissue specificity, as determined by thecapsid serotype and pseudotyping of AAV vectors to alter their tropismrange, will likely impact to their efficacy and use in therapy.

Serotype 2 (AAV2) has been the most extensively examined to date. AAV2presents a natural tropism towards skeletal muscles, neurons, vascularsmooth muscle cells, and hepatocytes. Three cell receptors have beendescribed for AAV2: heparan sulfate proteoglycan (HSPG), aVβ5 integrin,and fibroblast growth factor receptor 1 (FGFR-1). The first functions asa primary receptor, while the latter two have a co-receptor activity andenable AAV to enter the cell by receptor-mediated endocytosis.

Although AAV2 is the most popular serotype in various AAV-basedresearch, it has been shown that other serotypes can be more effectiveas gene delivery vectors. For instance AAV6 appears much better ininfecting airway epithelial cells, AAV7 presents very high transductionrate of murine skeletal muscle cells (similarly to AAV1 and AAV5), AAV8is superb in transducing hepatocytes, and AAV1 and 5 were shown to bevery efficient in gene delivery to vascular endothelial cells. In thebrain, most AAV serotypes show neuronal tropism, while AAV5 alsotransduces astrocytes. AAV6, a hybrid of AAV1 and AAV2, also shows lowerimmunogenicity than AAV2. Serotypes can differ with the respect to thereceptors they are bound to. For example AAV4 and AAV5 transduction canbe inhibited by soluble sialic acids (of different form for each ofthese serotypes), and AAV5 was shown to enter cells via theplatelet-derived growth factor receptor. Currently, rAAV8 and rAAV9 showthe most prominent features relevant to therapeutic use relative to allother serotypes and under undisturbed physiological conditions. (Gao, G,Zhong L, and Danos O (2011) Methods Mol. Biol. 807:93-118).

There are several steps in the AAV infection cycle, from infecting acell to producing new infectious particles. These are:

-   1. attachment to the cell membrane-   2. receptor-mediated endocytosis-   3. endosomal trafficking-   4. escape from the late endosome or lysosome-   5. translocation to the nucleus-   6. uncoating-   7. formation of double-stranded DNA replicative form of the AAV    genome-   8. expression of rep genes-   9. genome replication-   10. expression of cap genes, synthesis of progeny ssDNA particles-   11. assembly of complete virions, and-   12. release from the infected cell.

These steps may differ depending on the host cell type, which, in part,contributes to the defined and quite limited native tropism of AAV.Replication of the virus can also, even in regards to the same celltype, be dependent on the cell’s cycle phase at the time of infection.

The characteristic feature of the adeno-associated virus is a deficiencyin replication and thus, its inability to multiply in unaffected cells.The first factor described as providing successful generation of new AAVparticles was the adenovirus, from which the AAV name originated. It wasthen shown that AAV replication is facilitated by selected proteinsderived from the adenovirus genome, by other viruses such as HSV, or bygenotoxic agents, such as UV irradiation or hydroxyurea. The minimal setof the adenoviral genes required for efficient generation of progeny AAVparticles were discovered by Matsushita, Ellinger et al. (Matsushita T,Elliger S, Elliger C, et al. (1998) Gene Therapy 5: 938-45). Thisdiscovery paved the way for new production methods of recombinant AAV,which do not require adenoviral co-infection of the AAV-producing cells.In the absence of helper virus or genotoxic factors, AAV DNA can eitherintegrate into the host genome, or persist in episomal form. In theformer case integration is mediated by Rep78 and Rep68 proteins andrequires the presence of ITRs flanking the region being integrated. Inmice, the AAV genome has been observed persisting for long periods inquiescent tissues, such as skeletal muscles, in episomal form (acircular head-to-tail conformation).

Engineered Site-Specific Endonucleases. The present invention relates tothe use of rAAV vectors to deliver engineered, site-specificendonucleases. Site-specific, rare-cutting endonucleases can be used to“edit” the genomes of living cells or organisms by targeting adouble-stranded DNA break to a specific site in the genome that is thenrepaired by the cell’s DNA repair machinery. This process can oftenresult in DNA repair errors that, if they occur in the coding sequenceof a gene, can disrupt or frameshift the gene and thereby disable(knock-out) the gene. Alternatively, chromosomal DNA breaks are highlyrecombinigenic and, so, site-specific endonucleases can be used topromote homologous recombination between the chromosomal DNA sequenceand a transgenic sequence provided to the cell. This can result in, forexample, the targeted insertion of a transgene or the repair of a mutantgene that is responsible for disease.

Methods for producing engineered, site-specific endonucleases are knownin the art. For example, zinc-finger nucleases (ZFNs) can be engineeredto recognize and cut pre-determined sites in a genome. ZFNs are chimericproteins comprising a zinc finger DNA-binding domain fused to thenuclease domain of the FokI restriction enzyme. The zinc finger domaincan be redesigned through rational or experimental means to produce aprotein which binds to a pre-determined DNA sequence ~18 basepairs inlength. By fusing this engineered protein domain to the FokI nuclease,it is possible to target DNA breaks with genome-level specificity. ZFNshave been used extensively to target gene addition, removal, andsubstitution in a wide range of eukaryotic organisms (reviewed in DuraiS, et al. (2005) Nucleic Acids Res 33, 5978).

Likewise, TAL-effector nucleases (TALENs) can be generated to cleavespecific sites in genomic DNA. Like a ZFN, a TALEN comprises anengineered, site-specific DNA-binding domain fused to the FokI nucleasedomain (reviewed in Mak, et al. (2013) Curr Opin Struct Biol. 23:93-9).In this case, however, the DNA binding domain comprises a tandem arrayof TAL-effector domains, each of which specifically recognizes a singleDNA basepair. The large size of a TALEN makes it difficult to package inrAAV, limiting the utility of TALENs for compositions of the presentinvention comprising rAAV vectors. Thus, vectors created usinglentiviruses and/or retroviruses present an attractive candidate whenusing ZFNs and TALENs.

Compact TALENs are an alternative endonuclease architecture that avoidsthe need for dimerization (Beurdeley, et al. (2013) Nat Commun. 4:1762). A Compact TALEN comprises an engineered, site-specific TAL-effectorDNA-binding domain fused to the nuclease domain from the I-TevI homingendonuclease. Unlike FokI, I-TevI does not need to dimerize to produce adouble-strand DNA break so a Compact TALEN is functional as a monomer.Thus, it is possible to co-express two Compact TALENs in the same cell.Moreover, the Compact TALEN is smaller in size, making it much moreattractive in vector design. (Id.).

Engineered endonucleases based on the CRISPR/Cas9 system are also knownin the art (Ran, et al. (2013) Nat Protoc. 8:2281-2308; Mali et al.(2013) Nat Methods. 10:957-63). A CRISPR endonuclease comprises twocomponents: (1) a caspase effector nuclease, typically microbial Cas9;and (2) a short “guide RNA” comprising a ~20 nucleotide targetingsequence that directs the nuclease to a location of interest in thegenome. By expressing multiple guide RNAs in the same cell, each havinga different targeting sequence, it is possible to target DNA breakssimultaneously to multiple sites in in the genome. The primary drawbackof the CRISPR/Cas9 system is its reported high frequency of off-targetDNA breaks, which could limit the utility of the system for treatinghuman patients (Fu, et al. (2013) Nat Biotechnol. 31:822-6).

In the preferred embodiment of the invention, the DNA break-inducingagent is an engineered homing endonuclease (also called a“meganuclease”). Homing endonucleases are a group of naturally-occurringnucleases, which recognize 15-40 base-pair cleavage sites commonly foundin the genomes of plants and fungi. They are frequently associated withparasitic DNA elements, such as group 1 self-splicing introns andinteins. They naturally promote homologous recombination or geneinsertion at specific locations in the host genome by producing adouble-stranded break in the chromosome, which recruits the cellularDNA-repair machinery (Stoddard (2006) Q. Rev. Biophys. 38: 49-95).

Homing endonucleases are commonly grouped into four families: theLAGLIDADG family, the GIY-YIG family, the His-Cys box family and the HNHfamily. These families are characterized by structural motifs, whichaffect catalytic activity and recognition sequence. For instance,members of the LAGLIDADG family are characterized by having either oneor two copies of the conserved LAGLIDADG motif (see Chevalier et al.(2001) Nucleic Acids Res. 29(18): 3757-3774). The LAGLIDADG homingendonucleases with a single copy of the LAGLIDADG motif form homodimers,whereas members with two copies of the LAGLIDADG motif are found asmonomers.

I-CreI (SEQ ID NO: 1) is a member of the LAGLIDADG family of homingendonucleases, which recognizes and cuts a 22 basepair recognitionsequence in the chloroplast chromosome of the algae Chlamydomonasreinhardtii. Genetic selection techniques have been used to modify thewild-type I-CreI cleavage site preference (Sussman et al. (2004) J. Mol.Biol. 342: 31-41; Chames et al. (2005) Nucleic Acids Res. 33: e178;Seligman et al. (2002) Nucleic Acids Res. 30: 3870-9; Amould et al.(2006) J. Mol. Biol. 355: 443-58). More recently, a method ofrationally-designing mono-LAGLIDADG homing endonucleases capable ofcomprehensively redesigning I-CreI and other homing endonucleases totarget widely-divergent DNA sites, including sites in mammalian, yeast,plant, bacterial, and viral genomes has been described (WO 2007/047859).

As first described in WO 2009/059195, I-CreI and its engineeredderivatives are normally dimeric but can be fused into a singlepolypeptide using a short peptide linker that joins the C-terminus of afirst subunit to the N-terminus of a second subunit (Li, et al. (2009)Nucleic Acids Res. 37:1650-62; Grizot, et al. (2009) Nucleic Acids Res.37:5405-19.) Thus, a functional “single-chain” meganuclease can beexpressed from a single transcript. By delivering genes encoding twodifferent single-chain meganucleases to the same cell, it is possible tosimultaneously cut two different sites. This, coupled with the extremelylow frequency of off-target cutting observed with engineeredmeganucleases, makes them the preferred endonuclease for the presentinvention.

For many applications, it is necessary to deliver (a) gene(s) encodingengineered endonuclease(s) to the target cell or organism. For in vivoapplications, rAAV is a preferred delivery vector. However, rAAV vectorshave long persistence times in many cell types, particularlynon-dividing cells. Such persistence can activate immune response withinthe cell and cause disruption. Genome editing using engineeredendonucleases requires only a short burst of endonuclease expressionsuch that the endonuclease protein accumulates to a sufficientintracellular concentration to cut its recognition sequence in thegenome. Long-term expression of an endonuclease can result in unintendedoff-target DNA cutting or in an immune response directed toward cellsexpressing the foreign nuclease protein. Thus, there is a need for rAAVvectors encoding site-specific gene editing endonucleases in which thepersistence time of the vector is limited.

SUMMARY OF THE INVENTION

The present invention is a self-limiting rAAV vector having limitedpersistence time in a cell or organism due to the presence of arecognition sequence for a site-specific endonuclease within the vector.Thus, in one embodiment, the invention provides a general method forlimiting the persistence time of a vector. In another embodiment, theinvention provides self-limiting viral vectors with reduced persistencetime. In a third embodiment, the invention provides methods for usingself-limiting viral vectors for genome editing applications.

It is understood that any of the embodiments described below can becombined in any desired way, and any embodiment or combination ofembodiments can be applied to each of the aspects described below,unless the context indicates otherwise.

In some embodiments, the self-limiting viral vector is anadeno-associated virus (AAV) engineered to provide coding for apromoter, an endonuclease, and an endonuclease recognition site withinthe ITRs. The self-limiting viral vector delivers the endonuclease geneto a cell, tissue, or organism such that the endonuclease is expressedand able to cut the genome of the cell at a recognition site for theendonuclease endogenously within the genome. The delivered endonucleasewill also find its target site within the self-limiting viral vector,and cut the vector at this target site. Once cut, the 5′ and 3′ ends ofthe viral genome will be exposed and degraded by exonucleases, thuskilling the virus and ceasing production of the exogenous endonuclease.

In some aspects, the endonuclease of the self-limiting viral vector is ameganuclease, ZFN, TALEN, Compact TALEN, or CRISPR/Cas9, theendonuclease being either engineered or wild-type.

In more aspects, the location of the endonuclease recognition sequencemay vary within the self-limiting viral vector. In these aspects thegeneral construction of the self-limiting viral vector comprises from a5′ region of the ITRs: a promoter, a coding sequence for theendonuclease and poly A. The recognition sequence varies in itsposition. For example, in one aspect, the recognition sequence mayfollow, be 3′ from, the coding for the endonuclease and polyA.Alternatively, in other aspects, the recognition sequence may be placedupstream, or 5′ from, of the promoter in the self-limiting viral vector.Moreover, in some aspects, the recognition sequence may be placed withinan intron of the endonuclease sequence. Additionally, in another aspect,the endonuclease recognition sequence may be placed between the promoterand the endonuclease coding sequence. The endonuclease recognitionsequence may be placed within the self-limiting viral vector in anyposition where it will be recognized by the endonuclease and result intermination of transcription of the viral genome.

In some embodiments it is advantageous to modify the endonucleaserecognition sequence such that recognition by the endonuclease issub-optimal. In one aspect, an engineered meganuclease recognitionsequence may have 1-2 basepair changes selected from bases 1, 10, 11,12, 13, or 22 within the recognition sequence.

In some embodiments, the expressed endonuclease of the self-limitingviral vector is engineered to recognize a gene sequence of interest inthe host genome, such that the endonuclease recognizes and cuts thegenome at a specific site within the gene of interest, and wherein thecut initiates non-homologous end joining (NHEJ) such that mutations areintroduced into the gene, and the gene of interest can no longer betranslated correctly. In these embodiments, the endonuclease alsorecognizes the endonuclease recognition sequence within theself-limiting viral vector and cuts the vector genome, thus ceasing thetranscription of the endonuclease and the persistence of the viralvector.

In other embodiments, the self-limiting viral vector can remove a regionof interest within the cell genome. In these embodiments, theself-limiting viral vector contains coding for multiple endonucleasesand the endonuclease recognition sequence is placed between the 3′ endof one endonuclease sequence, and before the promoter of a secondendonuclease sequence, the endonuclease recognition site recognized byone of the expressed endonucleases, and each endonuclease identifyingand introducing a break in the genome of the cell at a site on eitherthe 5′ or 3′ end of a region of interest within the cell genome. Oncebroken, the region of interest being excised from the genome, and theresulting ends being religated in the genome, the region of interesthaving been removed from the cell genome.

In some aspects, where more than one endonuclease is contained withinthe self-limiting viral vector, the endonuclease recognition site may beplaced in variable places within the self-limiting viral vector that donot disrupt the functioning of the self-limiting viral vector, and therecognition site is specific to at least one of the endonucleases withinthe self-limiting viral vector. Moreover, the endonuclease recognitionsite capable of modification such that detection by the transcribedendonuclease is sub-optimal, as is described in other embodiments.

In other aspects, the self-limiting viral vector may be used to delivera transgene to a region of interest within the cell genome. In theseaspects, the self-limiting viral vector contains with in the ITRs from a5′ position: a promoter, endonuclease sequence, endonuclease recognitionsite, and a transgene flanked by homologous DNA sequences to the locusof interest. In further aspects, the expressed endonuclease recognizesand cuts a region of interest within the genome, and also recognizes andcuts at the endonuclease recognition site within the self-limiting viralvector. Furthermore, in these aspects, the 5′ end of the homologous DNAsequence adjacent to the transgene coded in the self-limiting viralvector is then integrated into the 5′ end of the region of interest andthe 3′ end of the homologous DNA sequence adjacent to the transgenecoded in the self-limiting viral vector is integrated into the 3; end ofthe region of interest by homologous recombination, thus providing thetransgene within the genome of the cell.

In other aspects, the self-limiting viral vector contains within theITRs from a 5′ position: a promoter, endonuclease sequence, endonucleaserecognition site, and a gene sequence. In these aspects, the expressedendonuclease cuts a gene sequence within the genome, and cuts at theendonuclease recognition site within the self-limiting viral vector.Furthermore, in these aspects, the 5′ end of gene sequence in theself-limiting viral vector is then integrated into the 5′ end of genesequence within the host cell genome containing a mutation and the 3′ ofthe gene sequence in the self-limiting viral vector integrated into the3′end of the gene sequence of the host cell genome by homologousrecombination.

In one aspect the rAAV containing the self-limiting viral vector isproduced using triple-transfection, wherein the packaging cell line istransfected with: 1. a plasmid containing “helper components,” 2. aplasmid containing the cap and rep genes, and 3. a plasmid containingthe self-limiting viral vector. In this aspect, the transfectedpackaging cell allowing for formation of the formed virus, that is thenpurified for infection of a cell, tissue, or organism.

In another aspect, the endonuclease coded for within the self-limitingviral vector is controlled by a tissue-specific promoter; the promoterinactive in the transfected packaging cell.

In further aspects, the self-limiting viral vector may be packaged incells not endogenously expressing endonuclease (i.e., the use ofmammalian promoters in microbial, plant, or insect cells).

The endonuclease gene of the self-limiting viral vector may also beoperably linked to an inducible promoter, thus introducing therequirement of a small molecule for expression in some embodiments.

In some embodiments, the rAAV particles may be produced in mammaliancell lines expressing a transcription repressor, preventing theexpression of the endonuclease gene within the self-limiting viralvector, wherein the transcription repressor can be encoded on a separatevector, the packaging vector outside of the ITRs, into the cap/repvector, or stably integrated into the genome of the packaging cell.

In another aspect, the self-limiting viral vectors may be used astherapeutic agents for the treatment of genetic disorders. In someembodiments, the self-limiting viral vectors may be delivered byintravenous injection, injected into tissues (e.g., intramuscular orsubretinal injection), hydrodynamic injection, intracranial injection,or direct injection.

In other embodiments, the self-limiting viral vectors are adenoviral orlentiviral/retroviral vectors to limit persistence times of the vectorin cells.

In one aspect, the invention provides a viral vector comprising: (a) afirst nucleic acid sequence encoding a first engineered nuclease; (b) afirst promoter operably linked to the first nucleic acid sequence,wherein the first promoter is positioned 5′ upstream of the firstnucleic acid sequence and drives expression of the first engineerednuclease in a target cell; and (c) a first vector recognition sequencewhich is recognized and cleaved by the first engineered nuclease.

In one embodiment, the viral vector can have a lower persistence time inthe target cell when compared to a viral vector which does not comprisethe recognition sequence but is otherwise identical.

In another embodiment, the recognition sequence can be identical to afirst chromosomal recognition sequence present in the genome of thetarget cell.

In another embodiment, the recognition sequence can be a sub-optimalrecognition sequence which is recognized and cleaved by the firstengineered nuclease. In some embodiments, the sub-optimal recognitionsequence differs by up to 2 nucleotides from an optimal recognitionsequence. In another such embodiment, wherein the sub-optimalrecognition sequence is a meganuclease recognition sequence, the 2differing nucleotides can be at base positions 1, 10, 11, 12, 13, or 22(from 5′ to 3′) of the sub-optimal recognition sequence.

In another embodiment, the first vector recognition sequence can bepositioned 5′ upstream of the first promoter.

In another embodiment, the first vector recognition sequence can bepositioned 3′ downstream of the first promoter and 5′ upstream of thefirst nucleic acid sequence.

In another embodiment, the first vector recognition sequence can bepositioned 3′ downstream of the first nucleic acid sequence.

In another embodiment, the first nucleic acid sequence can comprise,from 5′ to 3′, a first exon, an intron, and a second exon. In one suchembodiment, the first vector recognition sequence can be positionedwithin the intron of the first nucleic acid sequence. In a particularembodiment, the intron can be a human growth hormone intron (SEQ ID NO:2) or an SV40 large T antigen intron (SEQ ID NO: 3).

In another embodiment, the viral vector can further comprise a firstpolyA sequence positioned 3′ downstream of the first nucleic acidsequence. In one such embodiment, the first vector recognition sequencecan be positioned 3′ downstream of the first nucleic acid sequence and5′ upstream of the first polyA sequence. In another such embodiment, thefirst vector recognition sequence can be positioned 3′ downstream of thefirst polyA sequence.

In another embodiment, the viral vector can further comprise a transgenesequence.

In one such embodiment, the transgene sequence can be positioned 3′downstream of the first nucleic acid sequence. In another suchembodiment, the transgene sequence can be positioned 5′ upstream of thefirst promoter

In another such embodiment, the transgene sequence can be flanked bysequences homologous to sequences flanking a region of interest in thegenome of the target cell.

In another such embodiment, the first chromosomal recognition sequencecan be positioned within the region of interest in the genome of thetarget cell.

In another such embodiment, the first vector recognition sequence can bepositioned 5′ upstream of the transgene sequence.

In another such embodiment, the first vector recognition sequence can bepositioned 3′ downstream of the transgene sequence.

In another embodiment, the viral vector can further comprise a correctedgene.

In one such embodiment, the corrected gene sequence can be positioned 3′downstream of the first nucleic acid sequence.

In another such embodiment, the corrected gene sequence can bepositioned 5′ upstream of the first promoter.

In another such embodiment, the corrected gene sequence does notcomprise the first vector recognition sequence.

In another such embodiment, the corrected gene sequence can correspondto a mutated gene sequence present in the genome of the target cell,wherein the mutated gene sequence can differ from the corrected genesequence by at least one nucleotide and can comprise the firstchromosomal recognition sequence.

In another such embodiment, the first vector recognition sequence can bepositioned 5′ upstream of the corrected gene sequence.

In another such embodiment, the first vector recognition sequence can bepositioned 3′ downstream of the corrected gene sequence.

In another embodiment, the viral vector can further comprise a secondnucleic acid sequence encoding a second engineered nuclease.

In one such embodiment, the second nucleic acid sequence can bepositioned 5′ upstream of the first nucleic acid sequence, and 3′downstream of the first promoter, such that the first promoter drivesexpression of both the first engineered nuclease and the secondengineered nuclease.

In another such embodiment, the viral vector can further comprise asecond promoter operably linked to the second nucleic acid sequence,wherein the second promoter can be positioned 5′ upstream of the secondnucleic acid sequence and drives expression of the second engineerednuclease in the target cell.

In another such embodiment, the second promoter and the second nucleicacid sequence can be positioned 5′ upstream of the first promoter.

In another such embodiment, the second promoter and the second nucleicacid sequence can be positioned 3′ downstream of the first nucleic acidsequence.

In another such embodiment, the second engineered nuclease can recognizeand cleave a second chromosomal recognition sequence present in thegenome of the target cell.

In another such embodiment, the second promoter can be identical to thefirst promoter. In another such embodiment, the second promoter candiffer from the first promoter.

In another such embodiment, the first chromosomal recognition sequenceand the second chromosomal recognition sequence can flank the 5′ end andthe 3′ end of a region of interest in the genome of the target cell.

In another such embodiment, the first chromosomal recognition sequenceand the second chromosomal recognition sequence can be positioned on thesame chromosome.

In another such embodiment, the first chromosomal recognition sequenceand the second chromosomal recognition sequence can be positioned ondifferent chromosomes.

In another such embodiment, the first vector recognition sequence can bepositioned 5′ upstream of the second nucleic acid sequence.

In another such embodiment, the first vector recognition sequence can bepositioned 3′ downstream of the second promoter (if present) and 5′upstream of the second nucleic acid sequence.

In another such embodiment, the first vector recognition sequence can bepositioned 3′ downstream of the second nucleic acid sequence.

In another such embodiment, the second nucleic acid sequence cancomprise, from 5′ to 3′, a first exon, an intron, and a second exon. Inone such embodiment, the first vector recognition sequence can bepositioned within the intron of the second nucleic acid sequence. In aparticular embodiment, the intron can be a human growth hormone intron(SEQ ID NO: 2) or an SV40 large T antigen intron (SEQ ID NO: 3).

In another such embodiment, the viral vector can further comprise asecond polyA sequence positioned 3′ downstream of the second nucleicacid sequence. In one such embodiment, the first vector recognitionsequence can be positioned 3′ downstream of the second nucleic acidsequence and 5′ upstream of the second polyA sequence. In another suchembodiment, the first vector recognition sequence can be positioned 3′downstream of the second polyA sequence.

In another embodiment, the viral vector can be an adeno-associated virus(AAV) vector, a retroviral vector, a lentiviral vector, or an adenoviralvector. In various embodiments, the viral vector can be any viral vectorsuitable for use in the invention, including but not limited to, viralvectors of the families Adenoviridae, Baculoviridae, Hepadnaviridae,Herpesviridae, Iridoviridae, Papillomaviridae, Parvoviridae,Polyomaviridae, and Poxviridae.

In a particular embodiment, the viral vector can be an AAV vectorcomprising a 5′ inverted terminal repeat and a 3′ inverted terminalrepeat. In some such embodiments, the AAV vector can be asingle-stranded AAV vector or a self-complementary AAV vector.

In another embodiment, the first promoter can be a tissue-specificpromoter, a species-specific promoter, or an inducible promoter.

In another embodiment, the first engineered nuclease and/or the secondengineered nuclease can be an engineered meganuclease, a zinc fingernuclease (ZFN), a TALEN, a compact TALEN, or a CRISPR/Cas9. In aparticular embodiment, the first engineered nuclease and/or the secondengineered nuclease is an engineered meganuclease.

In another embodiment, the first promoter can comprise one or morebinding sites for a transcription repressor that binds to and silencesthe first promoter. In one such embodiment, the transcription repressorcan be a Tet repressor, a Lac repressor, a Cre repressor, or a Lambdarepressor.

In another embodiment, the first promoter can be an inducible promoter.In one such embodiment, the viral vector can further comprise a nucleicacid sequence encoding a ligand-inducible transcription factor whichregulates activation of the first promoter.

In all embodiments, it is understood that the viral vector can compriseonly one vector recognition sequence, but may comprise two or morevector recognition sequences which are recognized by the firstengineered nuclease and/or any additional engineered nucleases encodedby the viral vector.

In another aspect, the invention provides a recombinant DNA constructencoding any viral vector of the invention.

In one embodiment, the recombinant DNA construct can encode a viralvector wherein the first promoter comprises one or more binding sitesfor a transcription repressor that binds to and silences the firstpromoter. In one such embodiment, the recombinant DNA construct canfurther comprise a nucleic acid sequence encoding the transcriptionrepressor. In a particular embodiment, the nucleic acid sequenceencoding the transcription repressor can be positioned outside of thecoding sequence of the viral vector.

In another embodiment, the recombinant DNA construct can encode an AAVvector, a retroviral vector, a lentiviral vector, or an adenoviralvector. In a particular embodiment, the recombinant DNA constructencodes an AAV vector.

In another aspect, the invention provides a cell comprising anyrecombinant DNA construct of the invention.

In another aspect, the invention provides a method for producing a viralvector, the method comprising transforming a packaging cell with anyrecombinant DNA construct of the invention which encodes a viral vector,wherein the packaging cell produces the viral vector.

In one embodiment of the method, the viral vector can be a self-limitingviral vector which has a lower persistence time in the target cell whencompared to a control viral vector that is not self-limiting and doesnot comprise a recognition sequence for a first engineered nuclease.

In another embodiment of the method, the packaging cell can betransformed with a recombinant DNA construct of the invention whichcomprises a first promoter comprising one or more binding sites for atranscription repressor that binds to and silences the first promoter.In one such embodiment of the method, the recombinant DNA construct canfurther comprise a nucleic acid sequence encoding the transcriptionrepressor. In another such embodiment of the method, the nucleic acidsequence encoding the transcription repressor is positioned on therecombinant DNA construct outside of the coding sequence of the viralvector. In another such embodiment of the method, the packaging cell isfurther transformed with a second recombinant DNA construct comprising anucleic acid sequence encoding the transcription repressor. In anothersuch embodiment of the method, the packaging cell comprises in itsgenome a nucleic acid sequence encoding the transcription repressor,wherein the packaging cell stably expresses the transcription repressor.

In another embodiment of the method, the first promoter of therecombinant DNA construct can be a tissue-specific promoter that isinactive in the packaging cell; i.e., the tissue for which the promoterhas specificity differs from the tissue from which the packaging cell isderived.

In another embodiment of the method, the first promoter of therecombinant DNA construct can be a species-specific promoter that isinactive in the packaging cell; i.e., the species for which the promoterhas specificity differs from the species of the packaging cell. In onesuch embodiment of the method, the first promoter can be a mammalianpromoter and the packaging cell can be a microbial cell, an insect cell,or a plant cell.

In another such embodiment of the method, the packaging cell can be aninsect cell, and the first nucleic acid sequence encoding the firstengineered nuclease can comprise a mammalian intron that preventsexpression of the first engineered nuclease in the packaging cell.

In one particular embodiment of the method, the intron can be a humangrowth hormone intron (SEQ ID NO: 2) or an SV40 large T antigen intron(SEQ ID NO: 3).

In another embodiment of the method, the first promoter of therecombinant DNA construct can be an inducible-promoter which isregulated by a ligand-inducible transcription factor. In one suchembodiment of the method, the recombinant DNA construct can furthercomprise a nucleic acid sequence encoding the ligand-inducibletranscription factor. In a particular embodiment of the method, thenucleic acid sequence encoding the ligand-inducible transcription factorcan be positioned within the coding sequence of the viral vector.

In another embodiment of the method, the viral vector can be an AAVvector, a retroviral vector, a lentiviral vector, or an adenoviralvector.

In a particular embodiment of the method, the viral vector can be an AAVvector. In such an embodiment, the method can further comprisetransforming the packaging cell with: (a) a second recombinant DNAconstruct comprising a cap gene and a rep gene; and (b) a thirdrecombinant DNA construct comprising adenoviral helper components.

In another aspect, the invention provides a method for producing agenetically-modified eukaryotic cell by disrupting a target sequence ina chromosome of the eukaryotic cell. In such an aspect, the methodcomprises transducing the eukaryotic cell with any viral vector of theinvention; wherein the first engineered nuclease is expressed in theeukaryotic cell; wherein the first engineered nuclease produces a firstcleavage site at a first chromosomal recognition sequence positionedwithin the target sequence; and wherein the target sequence is disruptedby non-homologous end-joining at the first cleavage site, and whereinthe first engineered nuclease recognizes and cleaves the first vectorrecognition sequence in the viral vector..

In one embodiment of the method, the viral vector can have a lowerpersistence time in the target cell when compared to a viral vectorwhich does not comprise the recognition sequence, but is otherwiseidentical.

In another embodiment, the target sequence can comprise any gene ofinterest.

In another aspect, the invention provides a method for producing agenetically-modified eukaryotic cell including an exogenous sequence ofinterest inserted in a chromosome of the eukaryotic cell. In such anaspect, the method comprises: (a) transducing the eukaryotic cell withany viral vector of the invention, wherein the first engineered nucleaseencoded by the viral vector is expressed in the eukaryotic cell; and (b)introducing into the eukaryotic cell a nucleic acid comprising theexogenous sequence of interest; wherein the first engineered nucleaseproduces a first cleavage site in the chromosome at a first chromosomalrecognition sequence; and wherein the exogenous sequence of interest isinserted into the chromosome at the first cleavage site; and wherein thefirst engineered nuclease recognizes and cleaves the first vectorrecognition sequence in the viral vector.

In one embodiment of the method, the viral vector can have a lowerpersistence time in the target cell when compared to a viral vectorwhich does not comprise the recognition sequence but is otherwiseidentical.

In one embodiment of the method, the nucleic acid comprising theexogenous sequence of interest can further comprise sequences homologousto sequences flanking the first cleavage site, and the exogenoussequence of interest is inserted at the first cleavage site byhomologous recombination.

In another embodiment of the method, the nucleic acid comprising theexogenous sequence of interest can lack substantial homology tosequences flanking the first cleavage site, and the exogenous sequenceof interest can be inserted at the first cleavage site by non-homologousend-joining.

In another aspect, the invention provides a method for producing agenetically-modified eukaryotic cell including an exogenous sequence ofinterest (i.e., a transgene sequence) inserted in a chromosome of theeukaryotic cell. In such an aspect, the method comprises transducing theeukaryotic cell with any viral vector of the invention which comprises atransgene sequence; wherein the first engineered nuclease is expressedin the eukaryotic cell; and wherein the first engineered nucleaseproduces a first cleavage site in the chromosome at the firstchromosomal recognition sequence; and wherein the exogenous sequence ofinterest (i.e., the transgene sequence) is inserted into the chromosomeat the first cleavage site; and wherein the first engineered nucleaserecognizes and cleaves the first vector recognition sequence in theviral vector.

In one embodiment of the method, the viral vector can have a lowerpersistence time in the target cell when compared to a viral vectorwhich does not comprise the recognition sequence but is otherwiseidentical.

In another embodiment of the method, the exogenous sequence of interest(i.e., the transgene sequence) on the viral vector can be flanked bysequences homologous to sequences flanking the first cleavage site, andthe exogenous sequence of interest can be inserted at the first cleavagesite by homologous recombination.

In another embodiment of the method, the exogenous sequence of interest(i.e., the transgene sequence) on the viral vector can lack flankingsequences having substantial homology to the first cleavage site, andthe exogenous sequence of interest can be inserted at the first cleavagesite by non-homologous end-joining.

In another aspect, the invention provides a method for producing agenetically-modified eukaryotic cell including a corrected gene in achromosome of the eukaryotic cell. In such an aspect, the methodcomprises transducing the eukaryotic cell with any viral vector of theinvention comprising a corrected gene sequence; wherein the firstengineered nuclease is expressed in the eukaryotic cell; and wherein theeukaryotic cell comprises a mutated gene, wherein the mutated genecomprises the first chromosomal recognition sequence; and wherein thefirst engineered nuclease produces a first cleavage site at the firstchromosomal recognition sequence; and wherein the mutated gene isreplaced with the corrected gene sequence of the viral vector byhomologous recombination to produce a corrected gene in the eukaryoticcell; and wherein the first engineered nuclease recognizes and cleavesthe first vector recognition sequence in the viral vector.

In one embodiment of the method, the viral vector can have a lowerpersistence time in the target cell when compared to a viral vectorwhich does not comprise the recognition sequence but is otherwiseidentical.

In another embodiment of the method, the mutation present within themutated gene sequence can be positioned within the first chromosomalrecognition sequence. In another embodiment of the method, the mutationpresent within the mutated gene sequence can be positioned within 10bases, 100 bases, or up to 1000 bases of the first chromosomalrecognition sequence. Preferably, the mutation is positioned less than25 bases from the first chromosomal recognition sequence.

In another embodiment of the method, the corrected gene sequence in theviral vector does not comprise the first vector recognition sequence.

In another aspect, the invention provides a method for producing agenetically-modified eukaryotic cell including a deletion of a sequenceof interest in a chromosome of the eukaryotic cell. In such an aspect,the method comprises transducing the eukaryotic cell with any viralvector of the invention comprising a first nucleic acid sequenceencoding a first engineered nuclease and a second nucleic acid sequenceencoding a second engineered nuclease; wherein the first engineerednuclease and the second engineered nuclease are expressed in theeukaryotic cell; and wherein the first chromosomal recognition sequenceand the second chromosomal recognition sequence flank the sequence ofinterest on the chromosome; and wherein the first engineered nucleaseproduces a first cleavage site in the chromosome at the firstchromosomal recognition sequence; and wherein the second engineerednuclease produces a second cleavage site in the chromosome at the secondchromosomal recognition sequence; and wherein the intervening DNAfragment between the first cleavage site and the second cleavage site isexcised; and wherein the chromosome is repaired by re-ligation of thefirst cleavage site and the second cleavage site; and wherein the firstengineered nuclease recognizes and cleaves the first vector recognitionsequence in the viral vector.

In one embodiment of the method, the viral vector can have a lowerpersistence time in the target cell when compared to a viral vectorwhich does not comprise the recognition sequence but is otherwiseidentical.

In another aspect, the invention provides a method for producing agenetically-modified eukaryotic cell including an exogenous sequence ofinterest inserted in a chromosome of the eukaryotic cell. In such anaspect, the method comprises: (a) transducing the eukaryotic cell withany viral vector of the invention comprising a first nucleic acidsequence encoding a first engineered nuclease and a second nucleic acidsequence encoding a second engineered nuclease; wherein the firstengineered nuclease and the second engineered nuclease are expressed inthe eukaryotic cell; and (b) transforming the eukaryotic cell with anucleic acid comprising the exogenous sequence of interest; wherein thefirst engineered nuclease produces a first cleavage site in thechromosome at the first chromosomal recognition sequence; and whereinthe second engineered nuclease produces a second cleavage site in thechromosome at the second chromosomal recognition sequence; and whereinthe exogenous sequence of interest is inserted into the chromosomebetween the first cleavage site and the second cleavage site; andwherein the first engineered nuclease recognizes and cleaves the firstvector recognition sequence in the viral vector.

In one embodiment of the method, the viral vector can have a lowerpersistence time in the target cell when compared to a viral vectorwhich does not comprise the recognition sequence but is otherwiseidentical.

In another embodiment of the method, the exogenous sequence of interestcan be flanked on the nucleic acid by a sequence homologous to theregion 5′ upstream of the first cleavage site and a sequence homologousto the region 3′ downstream of the second cleavage site, and theexogenous sequence of interest can be inserted between the firstcleavage site and the second cleavage site by homologous recombination.

In another embodiment of the method, the nucleic acid comprising theexogenous sequence of interest can lack substantial homology tosequences flanking the first cleavage site and the second cleavage site,and the exogenous sequence of interest can be inserted into thechromosome between the first cleavage site and the second cleavage siteby non-homologous end-joining.

In another aspect, the invention provides a method for producing agenetically-modified eukaryotic cell by disrupting a first targetsequence and a second target sequence in the genome of the eukaryoticcell. In such an aspect, the method comprises transducing the eukaryoticcell with any viral vector of the invention comprising a first nucleicacid sequence encoding a first engineered nuclease and a second nucleicacid sequence encoding a second engineered nuclease; wherein the firstengineered nuclease and the second engineered nuclease are expressed inthe eukaryotic cell; and wherein the first engineered nuclease producesa first cleavage site at a first chromosomal recognition sequencepositioned within the first target sequence; and wherein the secondengineered nuclease produces a second cleavage site at a secondchromosomal recognition sequence positioned within the second targetsequence; and wherein the first target sequence and the second targetsequence are disrupted by non-homologous end-joining at the firstcleavage site and at the second cleavage site; and wherein the firstengineered nuclease recognizes and cleaves the first vector recognitionsequence in the viral vector.

In another embodiment of the method, the viral vector can have a lowerpersistence time in the target cell when compared to a viral vectorwhich does not comprise the recognition sequence but is otherwiseidentical.

In one embodiment of the method, the first target sequence and/or thesecond target sequence in the genome can be any gene of interest.

In another embodiment of the method, the first target sequence and thesecond target sequence can be on the same chromosome. In anotherembodiment of the method, the first target sequence and the secondtarget sequence can be on different chromosomes.

In another aspect, the invention provides a method for producing agenetically-modified eukaryotic cell including a first exogenoussequence of interest inserted in the genome of the eukaryotic cell. Insuch an aspect, the method comprises: (a) transducing the eukaryoticcell with any viral vector of the invention comprising a first nucleicacid sequence encoding a first engineered nuclease and a second nucleicacid sequence encoding a second engineered nuclease; and (b) introducinginto the eukaryotic cell a first nucleic acid comprising a firstexogenous sequence of interest; wherein the first engineered nucleaseand the second engineered nuclease are expressed in the eukaryotic cell;and wherein the first engineered nuclease produces a first cleavage siteat a first chromosomal recognition sequence positioned within a firsttarget sequence; and wherein the second engineered nuclease produces asecond cleavage site at a second chromosomal recognition sequencepositioned within a second target sequence; and wherein the firstexogenous sequence of interest is inserted into the genome at the firstcleavage site; and wherein the first engineered nuclease recognizes andcleaves the recognition sequence in the viral vector; and wherein thefirst engineered nuclease recognizes and cleaves the first vectorrecognition sequence in the viral vector.

In one embodiment of the method, the viral vector can have a lowerpersistence time in the target cell when compared to a viral vectorwhich does not comprise the recognition sequence but is otherwiseidentical.

In another embodiment of the method, the first target sequence can bedisrupted by insertion of the first exogenous sequence of interest intothe first cleavage site.

In another embodiment of the method, the first nucleic acid can comprisesequences flanking the first exogenous sequence of interest which arehomologous to sequences flanking the first cleavage site, and the firstexogenous sequence of interest can be inserted at the first cleavagesite by homologous recombination.

In another embodiment of the method, the first nucleic acid comprisingthe first exogenous sequence of interest can lack substantial homologyto sequences flanking the first cleavage site, and the first exogenoussequence of interest can be inserted into the first cleavage site bynon-homologous end-joining.

In another embodiment of the method, the second target sequence can bedisrupted by non-homologous end-joining following the production of thesecond cleavage site. Thus, in a particular embodiment, the first targetsequence can be disrupted following insertion of the first exogenoussequence of interest into the first cleavage site, and the second targetsequence can be disrupted by non-homologous end-joining following theproduction of the second cleavage site.

In another embodiment of the method, the first nucleic acid can comprisea first copy and a second copy of the first exogenous sequence ofinterest. In one such embodiment, the first copy can be inserted intothe genome at the first cleavage site and the second copy can beinserted into the genome at the second cleavage site. In such anembodiment, the first target sequence and the second target sequence caneach be disrupted by insertion of each copy of the first exogenoussequence of interest.

In one such embodiment of the method, the first nucleic acid cancomprise sequences flanking the first copy which are homologous tosequences flanking the first cleavage site, and/or can comprisesequences flanking the second copy which are homologous to sequencesflanking the second cleavage site. In such embodiments, the first copyand/or the second copy can be inserted at the first cleavage site and/orthe second cleavage site by homologous recombination.

In another such embodiment of the method, the first nucleic acidcomprising the first exogenous sequence of interest can lack substantialhomology to sequences flanking the first cleavage site and/or the secondcleavage site, and the first copy and/or the second copy can be insertedinto the first cleavage site and/or the second cleavage site bynon-homologous end-joining.

In another embodiment of the method, the first nucleic acid can compriseboth the first exogenous sequence of interest and a second exogenoussequence of interest. In one such embodiment of the method, the firstexogenous sequence of interest can be inserted into the first cleavagesite, and the second exogenous sequence of interest can be inserted intothe second cleavage site. In such an embodiment, the first targetsequence and/or the second target sequence can be disrupted followinginsertion of the first exogenous sequence of interest and/or the secondexogenous sequence of interest.

In one such embodiment of the method, the first nucleic acid cancomprise sequences flanking the first exogenous sequence of interestwhich are homologous to sequences flanking the first cleavage site. Thefirst nucleic acid can also comprise sequences flanking the secondexogenous sequence of interest which are homologous to sequencesflanking the second cleavage site. In such an embodiment, the firstexogenous sequence of interest can be inserted at the first cleavagesite and the second exogenous sequence of interest can be inserted atthe second cleavage site by homologous recombination.

In another such embodiment of the method, the first nucleic acidcomprising the first exogenous sequence of interest and the secondexogenous sequence of interest can lack substantial homology tosequences flanking the first cleavage site and/or the second cleavagesite, and the first exogenous sequence of interest can be inserted intothe first cleavage site, and the second exogenous sequence of interestcan be inserted into the second cleavage site, by non-homologousend-joining.

In another embodiment, the method can further comprise introducing intothe eukaryotic cell a second nucleic acid comprising a second exogenoussequence of interest.

In one such embodiment, the first exogenous sequence of interest can beinserted into the first cleavage site and the second exogenous sequenceof interest can be inserted into the second cleavage site. In aparticular embodiment, the first target sequence can be disrupted byinsertion of the first exogenous sequence of interest into the firstcleavage site, and the second target sequence can be disrupted byinsertion of the second exogenous sequence of interest into the secondcleavage site.

In another such embodiment of the method, the first nucleic acid cancomprise sequences flanking the first exogenous sequence of interestwhich are homologous to sequences flanking the first cleavage site,and/or the second nucleic acid can comprise sequences flanking thesecond exogenous sequence of interest which are homologous to sequencesflanking the second cleavage site. In such an embodiment, the firstexogenous sequence of interest can be inserted at the first cleavagesite, and/or the second exogenous sequence of interest can be insertedat the second cleavage site by homologous recombination.

In another such embodiment of the method, the first nucleic acidcomprising the first exogenous sequence of interest can lack substantialhomology to sequences flanking the first cleavage site, and/or thesecond nucleic acid comprising the second exogenous sequence of interestcan lack substantial homology to sequences flanking the second cleavagesite. In such an embodiment, the first exogenous sequence of interestcan be inserted into the first cleavage site, and/or the secondexogenous sequence of interest can be inserted in the second cleavagesite, by non-homologous end-joining.

In some such embodiments of the method, the first target sequence andthe second target sequence can be on the same chromosome. In other suchembodiments of the method, the first target sequence and the secondtarget sequence can be on different chromosomes.

In each embodiment of the method, the first target sequence and/or thesecond target sequence can be any gene of interest. In such embodiments,disruption of the first target sequence and/or disruption of the secondtarget sequence can result in reduced expression of the gene.

In another aspect, the invention provides a method for producing agenetically-modified non-human organism comprising: (a) producing agenetically-modified eukaryotic cell of the invention, wherein theeukaryotic cell is a non-human eukaryotic cell; and (b) growing thegenetically-modified non-human eukaryotic cell to produce thegenetically-modified non-human organism.

In one embodiment, the non-human eukaryotic cell is selected from thegroup consisting of a gamete, a zygote, a blastocyst cell, an embryonicstem cell, and a protoplast cell.

In another aspect, the invention provides a method for treating adisease in a subject in need thereof. In such an aspect, the methodcomprises administering to the subject a pharmaceutical compositioncomprising any viral vector of the invention and a pharmaceuticallyacceptable carrier.

In one embodiment, the disease can be cancer. In another embodiment, thedisease can be a genetic disorder. In such an embodiment, the diseasecan be a hereditary or a non-hereditary genetic disorder.

In another aspect, the invention provides a viral vector describedherein for use as a medicament. The invention further provides the useof a viral vector described herein in the manufacture of a medicamentfor treating a disease in a subject in need thereof.

In a particular aspect, the invention provides any AAV vector describedherein for use as a medicament. The invention further provides the useof an AAV vector in the manufacture of a medicament for treating adisease in a subject in need thereof.

In another aspect, the invention provides any genetically-modified cellof the invention for use as a medicament. The invention further providesthe use of any genetically-modified cell of the invention in themanufacture of a medicament for treating a disease in a subject in needthereof.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A- 1D. Diagrams of example self-limiting rAAV vectors. The vectorgenome is shown with ITRs at the ends. Each vector encodes asite-specific endonuclease and a recognition site for the endonucleasein the genome. FIGS. 1A-1D show example embodiments of vectorcompositions wherein the recognition site is positioned in variablelocations relative to the endonuclease gene and its associated promoter.

FIG. 2 . General method for using a self-limiting rAAV vector toknock-out a gene. In this embodiment, a self-limiting viral vector isdelivered to a cell or organism, the vector encoding a site-specificendonuclease that recognizes a target sequence in the coding sequence ofa gene of interest within the cell. Following infection with the vector,the cell expresses the endonuclease. The endonuclease then cuts both therecognition site in the genome of the cell and the recognition site inthe genome of the virus. The DNA break in the cell genome is repaired bynon-homologous end-joining (NHEJ), which introduces mutations into thegene, thus disabling it. The cut virus is quickly degraded byexonucleases and ceases to persist in the cell or organism.

FIG. 3 . General method for using a self-limiting rAAV to deliver a pairof endonucleases and excise a specific DNA fragment from the genome. Inthis embodiment, a self-limiting viral vector is delivered to a cell ororganism, the vector encoding a pair of site-specific endonucleases thatrecognize different target sequences in a locus of interest. Followinginfection with the vector, the cell or organism expresses theendonucleases. The endonucleases then cut both of the recognition sitesin the genome of the cell as well as a recognition site in the genome ofthe virus. The DNA breaks in the cell genome are repaired by deletingthe fragment intervening the breaks, followed by subsequent re-ligationof the genomic ends. The cut virus is quickly degraded by exonucleasesand ceases to persist in the cell or organism.

FIG. 4 . General method for using a self-limiting rAAV to insert atransgene at a defined location within the genome of a cell. In thisembodiment, a self-limiting viral vector is delivered to a cell ororganism, the vector encoding a site-specific endonuclease thatrecognizes a target sequence in a locus of interest within the cell ororganism’s genome. The self-limiting viral vector contains a transgeneflanked by a DNA sequence homologous to the locus of interest. Followinginfection with the vector, the cell expresses the endonuclease. Theendonucleases then cuts both the recognition site in the genome of thecell, as well as the recognition site in the genome of the virus. TheDNA break in the cell genome is repaired by homologous recombinationwith the viral vector, resulting in the insertion of the transgene atthe site of the DNA break. The cut virus is quickly degraded byexonucleases and ceases to persist in the cell or organism.

FIG. 5 . General method for using a self-limiting rAAV to repair amutation in the genome of a cell or organism. In this embodiment, aself-limiting viral vector is delivered to a cell or organism, thevector encoding a site-specific endonuclease that recognizes a targetsequence in the genome of the cell near a mutation associated withdisease. The self-limiting viral vector further comprises a partial“corrected” copy of the mutated gene. Following infection with thevector, the cell expresses the endonuclease. The endonucleases then cutsboth the recognition site in the genome of the cell as well as therecognition site in the genome of the virus. The DNA break in the cellgenome is repaired by homologous recombination with the viral vector,resulting in the “corrected” sequence being acquired by the genome ofthe cell. The cut virus is quickly degraded by exonucleases and ceasesto persist in the cell or organism.

FIGS. 6A - 6B. FIG. 6A) The OLR plasmid DNA vector (SEQ ID NO: 4) isillustrated. The OLR vector comprises a coding sequence for an MDX ½meganuclease operably-linked to a CMV promoter. The CMV promoter ismodified to include three Lac operon sequences. The OLR vector furthercomprises a cassette including a LacI coding sequence for expression ofa Lac repressor that binds to the Lac operons and suppresses expressionof the nuclease gene. FIG. 6B) The 3xOi plasmid DNA vector (SEQ ID NO:5) is illustrated. The 3xOi vector comprises the same elements as theOLR vector but lacks the cassette comprising the LacI coding sequence.Thus, no Lac repressor would be expressed and the nuclease gene wouldnot be suppressed.

FIG. 7 . Western blot for MDX ½ meganuclease expression. Experimentswere conducted to determine whether nuclease protein expression wassuppressed in viral packaging cells by using a Lac repressor. HEK293cells were mock electroporated, or electroporated with 2 µg of the OLRplasmid, the 3xOi plasmid, or a GFP (pMAX) RNA. At 1, 2, 4, 8, and 24hours post-transformation, cell lysates were prepared and nucleaseprotein expression was determined by Western blot analysis.

FIG. 8 . Vector map for the pDS GRK1 RHO½ L5-14 plasmid DNA vector (SEQID NO: 6). This vector comprises a RHO ½ meganuclease coding sequencethat is operably-linked to a GRK1 promoter, which is a tissue-specificpromoter that is specifically active in human retinal cells (i.e., rodcells). Thus, the meganuclease gene should not be expressed in viralpackaging cells such as HEK293 cells.

FIGS. 9A - 9B. Plasmid DNA vectors comprising introns within a nucleasecoding sequence. FIG. 9A) Vector map of the pDS CMV RHO ½ - HGH plasmidDNA vector (SEQ ID NO: 7). This vector comprises a RHO ½ meganucleasecoding sequence which includes a human growth hormone intron 1 (SEQ IDNO: 2) within its sequence. FIG. 9B) Vector map of the pDS CMV RHO ½ -SV40LT plasmid DNA vector (SEQ ID NO: 8). This vector comprises a RHO ½meganuclease coding sequence which includes an SV40 large T intron 1(SEQ ID NO: 2) within its sequence.

FIGS. 10A - 10B. FIG. 10A) Vector map of the pDS CMV 3xOi RHO ½ L514LacI plasmid DNA vector (SEQ ID NO: 9). FIG. 10B) Vector map of the pDSCMV 3xOi RHO ½ L514 plasmid DNA vector (SEQ ID NO: 10).

FIG. 11 . Western blot analysis of RHO ½ meganuclease protein expressionin transduced CHO cells. CHO cells were either mock transduced, ortransduced with the pDS CMV 3xOi RHO ½ L514 LacI vector (a self-limitingvector comprising a RHO ½ binding site) or the pDS CMV 3xOi RHO ½ L514vector (not self-limiting; no binding site). Protein was collected attime points of 2, 6, 12, 24, 48, and 72 hours and Western analysis wasperformed to detect RHO ½ meganuclease protein in cell lysates. The toppanel shows detection of RHO ½ meganuclease protein in cell lysates ateach time point. Lane 1 shows mock transduced cells. Lanes 2-7 show timepoints of 2, 6, 12, 24, 48, and 72 hours, respectively, for cellstransduced with the pDS CMV 3xOi RHO ½ L514 vector. Lanes 8-13 show timepoints of 2, 6, 12, 24, 48, and 72 hours, respectively, for cellstransduced with the self-limiting pDS CMV 3xOi RHO ½ L514 LacI vector.The arrow indicates the band associated with RHO ½ meganuclease protein.The bottom panel shows detection of a β-actin as a loading control.

FIG. 12 . PCR analysis of viral vector cleavage. CHO cells weretransduced with the self-limiting pDS CMV 3xOi RHO ½ L514 LacI vectorand DNA was isolated at 2, 6, 12, 24, 48, and 72 hourspost-transduction. An adapter molecule was designed having a 3′ overhangthat matches the 3′ overhang generated in the viral genome by the RHO ½meganuclease. PCR was then performed with adapter-ligated DNA and a pairof amplification primers, one matching the AAV sequence, and the othermatching the adapter molecule sequence. The resulting PCR products wereanalyzed on gel. In case of AAV digestion by RHO ½ and ligation toadapter, a PCR band with size 585 bps was expected to be observed.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 sets forth the amino acid sequence of the wild-type I-CreImeganuclease.

SEQ ID NO: 2 sets forth the nucleic acid sequence of the human growthhormone intron 1.

SEQ ID NO: 3 sets forth the nucleic acid sequence of the SV40 large Tantigen intron.

SEQ ID NO: 4 sets forth the nucleic acid sequence of the OLR DNAplasmid.

SEQ ID NO: 5 sets forth the nucleic acid sequence of the 3xOi plasmid.

SEQ ID NO: 6 sets forth the nucleic acid sequence of the pDS GRK1 RHO½L5-14 plasmid.

SEQ ID NO: 7 sets forth the nucleic acid sequence of the pDS CMV RHO ½-HGH plasmid.

SEQ ID NO: 8 sets forth the nucleic acid sequence of the pDS CMV RHO ½-SV40LT plasmid.

SEQ ID NO: 9 sets forth the nucleic acid sequence of the pDS CMV 3xOiRHO ½ L514 LacI plasmid.

SEQ ID NO: 10 sets forth the nucleic acid sequence of the pDS CMV 3xOiRHO ½ L514 plasmid.

DETAILED DESCRIPTION OF THE INVENTION 1.1 References and Definitions

The patent and scientific literature referred to herein establishesknowledge that is available to those of skill in the art. The entiredisclosures of the issued U.S. patents, pending applications, publishedforeign applications, and references, including GenBank databasesequences, that are cited herein are hereby incorporated by reference tothe same extent as if each was specifically and individually indicatedto be incorporated by reference.

Reference will now be made in detail to the preferred embodiments of theself-limiting viral vector, examples of which are illustrated in theaccompanying drawings.

As used herein, the term “cell” refers to a cell, whether it be part ofa cell line, tissue, or organism. “Cell” may refer to microbial, plant,insect, or animalian (mammalian, reptilian, avian, or otherwise) type,and where necessary, is specified.

As used herein, the term “meganuclease” refers to an endonuclease thatis derived from I-CreI. The term meganuclease, as used herein, refers toan engineered variant of I-CreI that has been modified relative tonatural I-CreI with respect to, for example, DNA-binding specificity,DNA cleavage activity, DNA-binding affinity, or dimerization properties.Methods for producing such modified variants of I-Crel are known in theart (i.e. WO 2007/047859). A meganuclease may bind to double-strandedDNA as a homodimer, as is the case for wild-type I-CreI, or it may bindto DNA as a heterodimer. A meganuclease may also be a “single-chainmeganuclease” in which a pair of DNA-binding domains derived from I-CreIare joined into a single polypeptide using a peptide linker.

As used herein, the term “single-chain meganuclease” refers to apolypeptide comprising a pair of meganuclease subunits joined by alinker. A single-chain meganuclease has the organization: N-terminalsubunit - Linker - C-terminal subunit. The two meganuclease subunits,each of which is derived from I-CreI, will generally be non-identical inamino acid sequence and will recognize non-identical DNA sequences.Thus, single-chain meganucleases typically cleave pseudo-palindromic ornon-palindromic recognition sequences. A single chain meganuclease maybe referred to as a “single-chain heterodimer” or “single-chainheterodimeric meganuclease” although it is not, in fact, dimeric. Forclarity, unless otherwise specified, the term “meganuclease” can referto a dimeric or single-chain meganuclease.

As used herein, the term “site specific endonuclease” means ameganuclease, TALEN, Compact TALEN, Zinc-Finger Nuclease, or CRISPR.

As used herein, the term “Compact TALEN” refers to an endonucleasecomprising a DNA-binding domain with 16-22 TAL domain repeats fused inany orientation to any portion of the I-TevI homing endonuclease.

As used herein, the term “Zinc-Finger Nuclease” refers to anendonuclease comprising a DNA-binding domain comprising 3-5 zinc-fingerdomains fused to any portion of the FokI nuclease domain.

As used herein, the term “TALEN” refers to an endonuclease comprising aDNA-binding domain comprising 16-22 TAL domain repeats fused to anyportion of the FokI nuclease domain.

As used herein, the term “CRISPR” refers to a caspase-based endonucleasecomprising a caspase, such as Cas9, and a guide RNA that directs DNAcleavage of the caspase by hybridizing to a recognition site in thegenomic DNA.

As used herein, with respect to a protein, the term “recombinant” meanshaving an altered amino acid sequence as a result of the application ofgenetic engineering techniques to nucleic acids which encode theprotein, and cells or organisms which express the protein. With respectto a nucleic acid, the term “recombinant” means having an alterednucleic acid sequence as a result of the application of geneticengineering techniques. Genetic engineering techniques include, but arenot limited to: PCR and DNA cloning technologies; transfection,transformation and other gene transfer technologies; homologousrecombination; site-directed mutagenesis; and gene fusion. In accordancewith this definition, a protein having an amino acid sequence identicalto a naturally-occurring protein, but produced by cloning and expressionin a heterologous host, is not considered recombinant. As used herein,the term “engineered” is synonymous with the term “recombinant.”

As used herein, the term “wild-type” refers to any naturally-occurringform of a meganuclease. The term “wild-type” is not intended to mean themost common allelic variant of the enzyme in nature but, rather, anyallelic variant found in nature. Wild-type homing endonucleases aredistinguished from recombinant or non-naturally-occurring meganucleases.

As used herein, the term “recognition sequence” refers to a DNA sequencethat is bound and cleaved by an endonuclease. In the case of ameganuclease, a recognition sequence comprises a pair of inverted, 9basepair “half sites” which are separated by four basepairs. In the caseof a single-chain meganuclease, the N-terminal domain of the proteincontacts a first half-site and the C-terminal domain of the proteincontacts a second half-site. Cleavage by a meganuclease produces fourbasepair 3′ “overhangs”. “Overhangs”, or “sticky ends” are short,single-stranded DNA segments that can be produced by endonucleasecleavage of a double-stranded DNA sequence. In the case of meganucleasesand single-chain meganucleases derived from I-CreI, the overhangcomprises bases 10-13 of the 22 basepair recognition sequence. In thecase of a Compact TALEN, the recognition sequence comprises a firstCNNNGN sequence that is recognized by the I-TevI domain, followed by anonspecific spacer 4-16 basepairs in length, followed by a secondsequence 16-22 bp in length that is recognized by the TAL-effectordomain (this sequence typically has a 5′ T base). Cleavage by a CompactTALEN produces two basepair 3′ overhangs. In the case of a CRISPR, therecognition sequence is the sequence, typically 16-24 basepairs, towhich the guide RNA binds to direct Cas9 cleavage. Cleavage by a CRISPRproduces blunt ends. In the case of a zinc finger, the DNA bindingdomains typically recognize an 18-bp recognition sequence comprising apair of nine basepair “half-sites” separated by 2-10 basepairs andcleavage by the nuclease creates a blunt end or a 5′ overhang ofvariable length (frequently four basepairs).

As used herein, the term “target site” or “target sequence” refers to aregion of the chromosomal DNA of a cell comprising a recognitionsequence for a site specific endonuclease.

As used herein, the term “homologous recombination” or “HR” refers tothe natural, cellular process in which a double-stranded DNA-break isrepaired using a homologous DNA sequence as the repair template (see,e.g. Cahill et al. (2006) Front. Biosci. 11:1958-1976). The homologousDNA sequence may be an endogenous chromosomal sequence or an exogenousnucleic acid that was delivered to the cell. The term “homology” is usedherein as equivalent to “sequence similarity” and is not intended torequire identity by descent or phylogenetic relatedness.

As used herein, the term “non-homologous end-joining” or “NHEJ” refersto the natural, cellular process in which a double-stranded DNA-break isrepaired by the direct joining of two non-homologous DNA segments (see,i.e. Cahill et al. (2006) Front. Biosci. 11:1958-1976). DNA repair bynon-homologous end-joining is error-prone and frequently results in theuntemplated addition or deletion of DNA sequences at the site of repair.The process of non-homologous end-joining occurs in both eukaryotes andprokaryotes such as bacteria.

As used herein, the term “re-ligation” refers to a process in which twoDNA ends produced by a pair of double-strand DNA breaks are covalentlyattached to one another with the loss of the intervening DNA sequencebut without the gain or loss of any additional DNA sequence. In the caseof a pair of DNA breaks produced with single-strand overhangs,re-ligation can proceed via annealing of complementary overhangsfollowed by covalent attachment of 5′ and 3′ ends by a DNA ligase.Re-ligation is distinguished from NHEJ in that it does not result in theuntemplated addition or removal of DNA from the site of repair.

As used herein, the term “concatamer” refers to long continuous DNAmolecules that contain multiple copies of the same DNA sequence linkedin series,

As used herein, the term “persistence” or “persist” refers to theviability of the self-limiting viral vector in the cell, tissue, ororganism of interest. Attenuating persistence time refers to thedegradation of the vector, and thus, viral genome.

As used herein, unless specifically indicated otherwise, the word “or”is used in the inclusive sense of “and/or” and not the exclusive senseof “either/or.”

2.1 Self-Limiting Viral Vectors

The present invention is based, in part, on the premise that a viralvector, such as a rAAV vector, will not persist in a cell after cleavageof the DNA by an endonuclease. rAAV is a preferred vector for deliveryof genome editing endonucleases to cells and tissues, but its longpersistence time in cells presents a problem. Genome editingapplications using site-specific endonucleases generally do not requirelong-term expression of the endonuclease gene, and long-term expressionmay be harmful. Long-term expression of endonucleases may hindercleavage specificity, thus introducing breakage in unintended sites,which may lead to detrimental consequences for cell health. Moreover,cell machinery is designed to detect and immunologically respond to theproduction of foreign proteins, such as endonucleases introduced by therAAV vector. (Mingozzi F, and High K (2013) Blood 122(1):23-26). Thus,the invention provides vectors in which vector persistence time is“self-limited” through a recognition sequence for the genome editingendonuclease already incorporated into the vector.

The self-limiting viral vector is thus able to deliver the endonucleasegene to a cell or tissue such that the endonuclease is expressed andable to modify the genome of the cell. In addition, the sameendonuclease will find its target site within the vector and will cutthe genome of the virus, exposing free 5′ and 3′ ends and initiatingdegradation by exonucleases. It is taught herein that cleavage of theviral genome will prevent the virus from forming concatamers that canpersist stably in the cell as episomes. Thus, the virus effectively“kills itself.”

The precise location of the endonuclease recognition sequence may vary,as exemplified in FIG. 1 . Certain configurations are preferred whenpossible. One preferred configuration is shown in FIG. 1C where theendonuclease recognition sequence is positioned within an intron in theendonuclease gene. Intracellular cleavage of such a vector is expectedto separate the two endonuclease exons such that the endonuclease genecan no longer be expressed. This leads to a more rapid attenuation ofendonuclease expression.. Alternatively, it is possible to position theendonuclease recognition sequence between the endonuclease gene and itspromoter (i.e. in the 5′ UTR), as shown in FIG. 1D. This configurationwill also quickly attenuate expression of the endonuclease while notadding as much additional size to the gene. Also depicted in FIG. 1A,the endonuclease recognition sequence may be placed after theendonuclease gene sequence and poly A, or the endonuclease recognitionsite may be placed before the promoter in the 5′ position of the ITR asdepicted in FIG. 1B. The endonuclease recognition site may be placed invarious locations, as long as the site does not interfere with theproper expression of the endonuclease, and is accessible by theexpressed nuclease.

The self-limiting viral vectors created can be used to infect cells,tissues, or organisms to achieve a multitude of therapeutic results. Forinstance, as exemplified in FIG. 2 , self-limiting viral vectors can beused to disable (“knock-out”) a gene. ]. In infected cells, theexpressed endonuclease within the cell recognizes a target sequencewithin a coding sequence of a gene of interest within the cell (thatcell being part of a cell line, tissue, or organism) and cuts the DNA.The DNA break in the cell’s genome will then be repaired bynon-homologous end-joining (NHEJ), such that mutations are introduced atthe target site that disables the gene of interest’s function.Subsequently, the expressed endonuclease recognizes and cuts theself-limiting viral vector at the endonuclease recognition sequence.Once cut, the self-limiting viral vector cannot produce concameters thatmay otherwise form and persist within the episomes. The self-limitingviral vector will cease to persist within the cell.

In other embodiments, described in FIG. 3 , the self-limiting viralvector comprises, starting at a 5′ position between the ITRs, apromoter, a first endonuclease coding sequence and poly A, anendonuclease recognitions site, a second promoter (which may be the sameas the first), a second endonuclease with polyA followed by the 3′ ITR.Notably, this configuration may be altered, specifically the location ofthe endonuclease recognition site (as discussed above and exemplified inFIG. 1 ). The endonuclease recognition site may be recognized by any oneof the endonucleases coded for within the self-limiting viral vector.FIG. 3 shows the endonuclease recognition site as being recognized bythe first endonuclease. Moreover, it is contemplated that more than twoendonucleases, each recognizing a unique site within a cell genome, maybe housed within the self-limiting viral vector. FIG. 3 depicts a vectorcoding for two endonucleases, which when expressed within an infectedcell, will each recognize their own target site, where the firstendonuclease recognizes a “site 1” and the second endonucleaserecognizes a “site 2.” The endonucleases cut the genome at each site,exposing the region of interest coded between each cut site. The regionof interest fragment is excised and degraded by cell machinery, and thecell genome is repaired by re-ligation. As stated above, the ligation ofthe genome is achieved by cell processes that maintain the integrity ofthe sequence and does not introduce additional sequence to the genome.The endonuclease subsequently recognizes the endonuclease recognitionsite within the self-limiting viral vector and cuts the viral genome.Once cut, the self-limiting viral vector cannot produce concatamers thatmay otherwise form and persist within the episomes. The self-limitingviral vector will cease to persist within the cell.

The self-limiting viral vectors of the present invention may be employedto introduce a new transgene into the infected cell’s genome. In theseembodiments, the self-limiting viral vector comprises from a 5′ positionbetween the ITRs: a promoter, endonuclease coding sequence and poly A,an endonuclease recognition site, a homologous DNA sequence, thetransgene, and another homologous sequence at the 3′ position within theITRs. Notably, this configuration may be altered, specifically thelocation of the endonuclease recognition site (as discussed above andexemplified in FIG. 1 ). For example, the endonuclease site could bepositioned on the 3′ end of the homologous DNA sequence of thetransgene, or in an intron contained within the transgene sequence.After infection, the endonuclease is expressed within the cell, andrecognizes a site within a region of interest. The endonuclease cleavesa site within the gene of interest, exposing 5′ and 3′ ends of the cellgenome. The self-limiting viral vector contains matching homologous DNAfor the exposed 5′ and 3′ ends of the cleaved cell genome. By homologousrecombination, the homologous DNA regions flanking the 5′ and 3′ ends ofthe transgene recombine with the cleaved portion of the cell genome andthe transgene of the self-limiting viral vector is inserted within thecell genome. The endonuclease subsequently recognizes the endonucleaserecognition site within the self-limiting viral vector and cuts theviral genome. Once cut, the self-limiting viral vector cannot produceconcameters that may otherwise form and persist within the episomes. Theself-limiting viral vector will cease to persist within the cell.

As shown in FIG. 5 , the self-limiting viral vectors of the presentinvention may be dispatched to alter a gene sequence within a cellgenome (as previously defined, that cell being part of a cell line,tissue, or organism). In these embodiments, the self-limiting viralvector comprises at a 5′ position of the ITRs, a promoter, anendonuclease coding sequence with poly A, an endonuclease recognitionsite, a gene sequence and the 3′ position within the ITRs. The expressedendonuclease recognizes and cuts a site within the gene sequence in theinfected cell’s genome. This cut exposes 5′ and 3′ ends of the infectedcell’s genome that can recombine with the 5′ and 3′ ends of the genesequence encoded within the self-limiting viral vector. The genesequence is inserted into the infected cell’s genome through homologousrecombination. The endonuclease subsequently recognizes the endonucleaserecognition site within the self-limiting viral vector and cuts theviral genome. Once cut, the self-limiting viral vector cannot produceconcameters that may otherwise form and persist within the episomes. Theself-limiting viral vector will cease to persist within the cell

2.2 Kinetic Balancing

In some cases, it may be advantageous to modify the recognition sequencein the self-limiting viral vector to make it sub-optimal. The viralvector should not be cut before a sufficient concentration ofendonuclease has been accumulated in the cell to modify the cell’sgenome in the desired manner. Because the chromosomal target sequence ofinterest will be chromatainized, it is more difficult to access than anepisomal vector sequence. Thus, higher concentrations of endonucleaseare likely required to cut the chromosomal recognition site in thegenome of the cell. If the transcribed endonuclease attacks therecognition sequence in the self-limiting viral vector before theappropriate amount of endonuclease is achieved, recognition of thetarget site within the cell may be unrealized. The use of sub-optimalrecognition sequences in the viral vector is “kinetic balancing,”because it is done to coordinate the timing of DNA cleavage such thatthe genome of the cell is cut first, followed by the genome of thevirus.

In general, sub-optimal recognition sequences can be generated bydeviating from the sequence that the endonuclease was engineered torecognize. An engineered meganuclease, for example, recognizes a 22 bpsequence but will tolerate certain 1-2 basepair changes in its preferredsequence. These modified sequences are typically cut less efficientlythan the preferred sequence and, so, are suitable for incorporation intoself-limiting viral vectors. In selecting a sub-optimal recognitionsequence for incorporation into self-limiting viral vectors, it iscritical that the sub-optimal site is still cut by the nuclease, albeitless efficiently than the preferred sequence. For each of the engineeredendonuclease types, regions of a recognition sequence may be able totolerate changes. For example, engineered meganucleases toleratesingle-base changes at bases 1, 10, 11, 12, 13, and 22 of therecognition sequence (Jurica MS, Monnat RJ Jr, Stoddard BL (1998) Mol.Cell. 2(4): 469-76).

Experimental methods to evaluate and quantify site-specific DNA cleavagemay be performed, including in vitro DNA digests with purifiedendonuclease protein and cell-based reporter assays (Chevalier B, TurmelM, Lemieux C, Monnat RJ Jr, Stoddard BL (2003) J. Mol. Biol. 329(2):253-69). These methods can be used to evaluate a variety of sub-optimalrecognition sequences to determine the sequences that are cut lessefficiently than the preferred recognition sequence in the genome of thecell.

2.3 Methods for Producing Self-Limiting Viruses

rAAV virus is typically produced in mammalian cell lines such asHEK-293. Because the viral cap and rep genes are removed from the vectorto prevent its self-replication to make room for the therapeutic gene(s)to be delivered (e.g. the endonuclease gene), it is necessary to providethese in trans in the packaging cell line. In addition, it is necessaryto provide the “helper” (e.g. adenoviral) components necessary tosupport replication (Cots D, Bosch A, Chillon M (2013) Curr. Gene Ther.13(5): 370-81). Frequently, rAAV is produced using a triple-transfectionin which a cell line is transfected with a first plasmid encoding the“helper” components, a second plasmid comprising the cap and rep genes,and a third plasmid comprising the viral ITRs containing the interveningDNA sequence to be packaged into the virus. Viral particles comprising agenome (ITRs and intervening gene(s) of interest) encased in a capsidare then isolated from cells by freeze-thaw cycles, sonication,detergent, or other means known in the art. Particles are then purifiedusing cesium-chloride density gradient centrifugation or affinitychromatography and subsequently delivered to the gene(s) of interest tocells, tissues, or an organism such as a human patient.

Because rAAV particles are typically produced (manufactured) in cells,precautions must be taken in practicing the current invention to ensurethat the site-specific endonuclease is NOT expressed in the packagingcells. Because the viral genomes of the invention comprise a recognitionsequence for the endonuclease, any endonuclease expressed in thepackaging cell line will be capable of cleaving the viral genome beforeit can be packaged into viral particles. This will result in reducedpackaging efficiency and/or the packaging of fragmented genomes. Severalapproaches can be used to prevent endonuclease expression in thepackaging cells, including:

1. The endonuclease can be placed under the control of a tissue-specificpromoter that is not active in the packaging cells. For example, if aself-limiting viral vector is developed for delivery of (an)endonuclease gene(s) to muscle tissue, a muscle-specific promoter can beused. Examples of muscle-specific promoters include C5-12 (Liu, et al.(2004) Hum Gene Ther. 15:783-92), the muscle-specific creatine kinase(MCK) promoter (Yuasa, et al. (2002) Gene Ther. 9: 1576-88), or thesmooth muscle 22 (SM22) promoter (Haase, et al. (2013) BMC Biotechnol.13:49-54). Examples of CNS (neuron)-specific promoters include the NSE,Synapsin, and MeCP2 promoters (Lentz, et al. (2012) Neurobiol Dis.48:179-88). Examples of liver-specific promoters include albuminpromoters ( such as Palb), human α1-antitrypsin (such as Pal AT), andhemopexin (such as Phpx) (Kramer, MG et al., (2003) Mol. Therapy7:375-85). Examples of eye-specific promoters include opsin, and cornealepithelium-specific K12 promoters (Martin KRG, Klein RL, and Quigley HA(2002) Methods (28): 267-75) (Tong Y, et al., (2007) J Gene Med,9:956-66). These promoters, or other tissue-specific promoters known inthe art, are not highly-active in HEK-293 cells and, thus, will notexpected to yield significant levels of endonuclease gene expression inpackaging cells when incorporated into self-limiting viral vectors ofthe present invention. Similarly, the self-limiting viral vectors of thepresent invention contemplate the use of other cell lines with the useof incompatible tissue specific promoters (i.e., the HeLa cell line(human epithelial cell) and using the liver-specific hemopexinpromoter). Other examples of tissue specific promoters include: synovialsarcomas PDZD4 (cerebellum), C6 (liver), ASB5 (muscle), PPP1R12B(heart), SLC5A12 (kidney), cholesterol regulation APOM (liver), ADPRHL1(heart), and monogenic malformation syndromes TP73L (muscle). (Jacox E,et al., (2010) PLoS One v.5(8):e12274).

2. Alternatively, the vector can be packaged in cells from a differentspecies in which the endonuclease is not likely to be expressed. Forexample, viral particles can be produced in microbial, insect, or plantcells using mammalian promoters, such as the cytomegalovirus- or SV40virus-early promoters, which are not active in the non-mammalianpackaging cells. In a preferred embodiment, viral particles are producedin insect cells using the baculovirus system as described by Gao, et al.(Gao, H., et al. (2007) J. Biotechnol. 131(2):138-43). An endonucleaseunder the control of a mammalian promoter is unlikely to be expressed inthese cells (Airenne, KJ, et al. (2013) Mol. Ther. 21(4):739-49).Moreover, insect cells utilize different mRNA splicing motifs thanmammalian cells. Thus, it is possible to incorporate a mammalian intron,such as the human growth hormone (HGH) intron (SEQ ID NO: 2), or theSV40 large T antigen intron (SEQ ID NO:3), into the coding sequence ofan endonuclease (see, for example, FIG. 1C). Because these introns arenot spliced efficiently from pre-mRNA transcripts in insect cells,insect cells will not express a functional endonuclease and will packagethe full-length genome. In contrast, mammalian cells to which theresulting rAAV particles are delivered will properly splice the pre-mRNAand will express functional endonuclease protein. Haifeng Chen hasreported the use of the HGH and SV40 large T antigen introns toattenuate expression of the toxic proteins barnase and diphtheria toxinfragment A in insect packaging cells, enabling the production of rAAVvectors carrying these toxin genes (Chen, H (2012) Mol Ther NucleicAcids. 1(11): e57).

3. The endonuclease gene can be operably linked to an inducible promotersuch that a small-molecule inducer is required for endonucleaseexpression. Examples of inducible promoters include the Tet-On system(Clontech; Chen H., et al., (2015) BMC Biotechnol. 15(1):4)) and theRheoSwitch system (Intrexon; Sowa G., et al., (2011) Spine, 36(10):E623-8). Both systems, as well as similar systems known in the art, relyon ligand-inducible transcription factors (variants of the Tet Repressorand Ecdysone receptor, respectively) that activate transcription inresponse to a small-molecule activator (Doxycycline or Ecdysone,respectively). Practicing the current invention using suchligand-inducible transcription activators includes: 1) placing theendonuclease gene under the control of a promoter that responds to thecorresponding transcription factor, the endonuclease gene having (a)binding site(s) for the transcription factor; and 2) including the geneencoding the transcription factor in the packaged viral genome. Thelatter step is necessary because the endonuclease will not be expressedin the target cells or tissues following rAAV delivery if thetranscription activator is not also provided to the same cells. Thetranscription activator then induces endonuclease gene expression onlyin cells or tissues that are treated with the cognate small-moleculeactivator. This approach is advantageous because it enables endonucleasegene expression to be regulated in a spatio-temporal manner by selectingwhen and to which tissues the small-molecule inducer is delivered.However, the requirement to include the inducer in the viral genome,which has significantly limited carrying capacity, creates a drawback tothis approach.

4. In another preferred embodiment, rAAV particles are produced in amammalian cell line that expresses a transcription repressor thatprevents expression of the endonuclease. Transcription repressors areknown in the art and include the Tet-Repressor, the Lac-Repressor, theCro repressor, and the Lambda-repressor. Many nuclear hormone receptorssuch as the ecdysone receptor also act as transcription repressors inthe absence of their cognate hormone ligand. To practice the currentinvention, packaging cells are transfected/transduced with a vectorencoding a transcription repressor and the endonuclease gene in theviral genome (packaging vector) is operably linked to a promoter that ismodified to comprise binding sites for the repressor such that therepressor silences the promoter. The gene encoding the transcriptionrepressor can be placed in a variety of positions. It can be encoded ona separate vector; it can be incorporated into the packaging vectoroutside of the ITR sequences; it can be incorporated into the cap/repvector or the adenoviral helper vector; or, most preferably, it can bestably integrated into the genome of the packaging cell such that it isexpressed constitutively. Some methods to modify common mammalianpromoters to incorporate transcription repressor sites have beendisclosed in the art. For example, Chang and Roninson modified thestrong, constitutive CMV and RSV promoters to comprise operators for theLac repressor and showed that gene expression from the modifiedpromoters was greatly attenuated in cells expressing the repressor(Chang BD, and Roninson IB (1996) Gene 183:137-42). The use of anon-human transcription repressor ensures that transcription of theendonuclease gene will be repressed only in the packaging cellsexpressing the repressor and not in target cells or tissues transducedwith the resulting self-limiting rAAV vector.

2.4 Methods for Delivering Self-Limiting Viral Vectors to Human Patientsand Animals

The self-limiting viral vectors of the invention, with their significantsafety advantages relative to conventional gene-therapy vectors, will beused as therapeutic agents for the treatment of genetic disorders. Fortherapeutic applications, route of administration is an importantconsideration. These self-limiting viral vectors may be deliveredsystemically via intravenous injection, especially where the targettissues for the therapeutic are liver (e.g. hepatocytes) or vascularepithelium/endothelium. Alternatively, the self-limiting viral vectorsof the invention may be injected directly into target tissues. Forexample, rAAV can be delivered to muscle cells via intramuscularinjection (Maltzahn, et al. (2012) Proc Natl Acad Sci USA.109:20614-9),or hydrodynamic injection (Taniyama, et al. (2012) Curr Top Med Chem.12:1630-7 and Hegge, et al. (2010) Hum Gene Ther. 21:829-42). Deliveryto CNS can be accomplished by systemic delivery or intracranialinjection (Weinberg, et al. (2013) Neuropharmacology. 69:82-8, Bourdenx,et al. (2014) Front MolNeurosci.7:50, and Ojala DS, et al. (2015)Neuroscientist. 21(1):84-98). Direct injection (e.g. subretinalinjection) is the preferred route of administration for the eye (WillettK and Bennett J (2013) Front Immunol. 4:261 and Colella P and AuricchioA (2012) Hum Gene Ther. 23(8):796-807.)

2.5 Self-Limiting Adenoviral and Retroviral Vectors

While the preferred embodiments of the invention are self-limiting rAAVvectors, the same principles can be applied to adenoviral andlentiviral/retroviral vectors to limit the persistence times of thesevectors in cells. These viral vectors have significantly larger genomesand, hence, larger “carrying capacities” than AAV which makes thempreferable for the delivery of larger gene payloads to the cell. Indeed,for applications involving the use of a gene editing endonuclease toinsert a transgene into the genome (as in FIG. 4 ), adenoviral orlentiviral/retroviral vectors are preferred when the transgene is largerthan ~3.5 kb. For other applications (e.g. FIGS. 2, 3, and 5 )adenoviral or lentiviral/retroviral vectors are preferred when the geneediting endonuclease is too large to be encoded by rAAV. This isparticularly applicable when employing TALENs and most CRISPR/Cas9endonucleases.

Adenovirus and lentiviruses/retroviruses naturally integrate into thegenome of the host cell. To be useful for the present invention, theability of the virus to integrate into the genome must be attenuated.For lentiviral/retroviral vectors, this is accomplished by mutating theint gene encoding the virus integrase. For example, Bobis-Wozowicz, etal. used an integration-deficient retroviral vector to deliverzinc-finger nucleases to human and mouse cells (Bobis-Wozowicz, et al(2014) Nature Scientific Reports 4:4656) (Qasim W, Vink CA, Thrasher AJ(2010) Mol. Ther. 18(7):1263-67), (Wanisch K, Yáñez-Muñoz RJ (2009) Mol.Ther. 17(8): 1316-32), (Nowrouzi A, et al. (2011) Viruses 3(5):429-55).

EXAMPLES

This invention is further illustrated by the following examples, whichshould not be construed as limiting. Those skilled in the art willrecognize, or be able to ascertain, using no more than routineexperimentation, numerous equivalents to the specific substances andprocedures described herein. Such equivalents are intended to beencompassed in the scope of the claims that follow the examples below.

Example 1 Promoter Silencing in Viral Packaging Cells 1. Rationale

In one example of the self-limiting system described herein, a nucleaseis expressed from its gene on a viral vector. The nuclease thenrecognizes and cleaves a recognition sequence integrated within theviral vector at any number of positions. In some examples, therecognition sequence can be positioned within an intron that isintegrated into the nuclease gene sequence. As a result, the viralvector is cleaved and degraded by the cell in a self-limiting manner.

A major obstacle to utilizing this concept to produce a packaged virus,such an as AAV virus, is preventing the nuclease from being expressed inthe packaging cell line during the production of the viral vector. Ifexpressed in the packaging cell line, the nuclease could prematurely cutthe viral vectors, as well as the plasmid DNA encoding the viralvectors, preventing packaging of whole, intact genomes that contain anintact meganuclease gene. Thus, one goal of the present studies was todetermine strategies for turning off expression of the nuclease in thepackaging cell line, but allowing the nuclease to be expressed in cellstransfected with the self-limiting virus.

2. Recombinant DNA Plasmids Comprising a Lac Repressor

In one approach, recombinant DNA plasmids were produced which compriseda Lac operator-repressor system. As reported by Cronin et al., three Lacoperators within a mammalian promoter can inhibit transcription bybinding Lac repressor and inhibiting RNA polymerase from processingthrough (Cronin, C.A., Gluba, W., and Scrable, H. (2001) Genes Dev.15(12): 1506 - 1517). It was reasoned that placing three Lac operatorsbetween a CMV promoter and a nuclease gene would allow similarinhibition of expression in the presence of a Lac repressor.

Thus, a DNA plasmid was produced which comprised a CMV promoteroperably-linked to a nuclease gene encoding a meganuclease referred toas MDX ½. This plasmid, referred to herein as the OLR plasmid, isillustrated in FIG. 6A, and its nucleic acid sequence is set forth inSEQ ID NO: 4. As shown, the CMV promoter was modified to include threeLac operators. The naturally-occurring Lac operator sequences weresubstituted with ideal operator sequences because the Lac repressorbinds more tightly to the ideal operator and, hence, should causestronger resistance to the RNA polymerase and better repression.Further, a mammalian expression cassette was built for the Lac repressorconsisting of a CMV promoter and SV40 poly A. The LacI gene was notcodon-optimized for Lac repressor. The Lac repressor expression cassettewas integrated into the plasmid backbone outside of the viral invertedterminal repeats (ITRs). This positioning was very important, as itallowed for expression of the Lac repressor in the packaging cell lineto prevent expression of the meganuclease, but because the Lac repressorexpression cassette is not packaged in the AAV virus, there should be norepression of the meganuclease in the target cells transfected with theAAV virus.

Additionally, a second DNA plasmid was produced which comprised a CMVpromoter operably-linked to a nuclease gene encoding the MDX ½meganuclease. This plasmid, referred to herein as the 3xOi plasmid, isillustrated in FIG. 6B, and its nucleic acid sequence is set forth inSEQ ID NO: 5. As shown, the CMV promoter in the 3xOi plasmid was alsomodified to include three Lac operators, but the 3xOi plasmid did notcomprise the LacI cassette to express the Lac repressor. Thus, it wasexpected that there would be no suppression of meganuclease expressionin the viral packaging cells.

3. Nuclease Protein Expression in HEK293 Cells

Experiments were conducted to determine whether nuclease proteinexpression was suppressed in viral packaging cells by using a Lacrepressor. HEK293 cells were mock electroporated, or electroporated with2 µg of the OLR plasmid, the 3xOi plasmid, or a GFP (pMAX) RNA using aBioRad Gene Pulser Xcell according to the manufacturer’s instructions.At 1, 2, 4, 8, and 24 hours post-transformation, cell lysates wereprepared by removing media, washing cells with phosphate bufferedsaline, adding 150 µL of RIPA buffer, scraping cells from their wells,and freezing lysates at -80° C. Lysates from mock electroporated cellswere obtained at 24 hours.

Nuclease protein expression was determined by Western blot analysis.Cell lysates were incubated at -80° C. for 15 minutes, thawed,re-frozen, re-thawed, and then centrifuged for 10 minutes at 16,000xg.Supernatants were collected and protein concentrations were determinedby the BCA method in combination with a plate reader. 9 µg total proteinper sample was electrophoresed under reducing conditions on a 12-well4-12% polyacrylamide gel. Duplicate gels were also run. Proteins weretransferred to PVDF membranes and blocked for 3 hours in TBST (0.1%) and5% milk.

To stain for the meganuclease, one membrane was incubated with a rabbitanti-I-CRE antibody (1:8000) in TBST (0.1%) and 5% milk overnight (~18hours) at 4° C. The membrane was washed 6 times for 5 minutes in TBST(0.1%). The membrane was then incubated with a peroxidase-labeled goatanti-rabbit antibody at 1:40k in TBST (0.1%) with 5% milk for 60 minutesat room temperature. The membrane was then washed 6 times for 5 minutesin TBST (0.1%) and incubated for 5 minutes in Amersham’s ECL PrimeWestern Blotting Detection Reagent. The membrane was developed onKodak’s BioMax XAR film.

As a control, the second membrane was stained for β-actin. The secondmembrane was incubated with a monoclonal anti β-actin antibody(1:15,000) in TBST (0.1%) and 5% milk overnight (~18 hrs) at 4° C. Themembrane was washed 6 times for 5 minutes in TBST (0.1%). The membranewas then incubated with a peroxidase-labeled goat antimouse antibody at1:40k in TBST (0.1%) with 5% milk for 60 minutes at room temperature.The membrane was then washed 6 times for 5 minutes in TBST (0.1%) andincubated for 5 minutes in Amersham’s ECL Prime Western BlottingDetection Reagent. The membrane was developed on Kodak’s BioMax XARfilm.

The Western blot analysis showed that cells transformed with the 3xOiplasmid, which did not comprise the Lac repressor sequence, showed anincrease in MDX ½ protein expression throughout the course of theexperiment (FIG. 7 ). By contrast, expression of MDX ½ protein wasdramatically suppressed over 24 hours in HEK293 cells transformed withthe OLR plasmid encoding the Lac repressor. Indeed, even at 24 hours,only a very small fraction of the MDX ½ meganuclease produced by the3xOi plasmid can be seen in cells transformed with the OLR plasmid.

4. Conclusion

It is clear from this study that the incorporation of a repressorsequence (such as the Lac repressor) into the DNA plasmid, as well asoperators for the repressor in the promoter operably-linked to thenuclease gene, can effectively silence expression of a nuclease in aviral packaging cell line, such as HEK293.

Example 2 Suppression of Nuclease Expression in Viral Packaging CellsUsing a Tissue-Specific Promoter 1. Rationale

In another example, nuclease expression can be suppressed in viralpackaging cells by the use of a tissue-specific promoter that isoperably-linked to the nuclease gene. In this approach, thetissue-specific promoter is not active in the viral packaging cell butis active in the target cell into which the viral vector is ultimatelytransduced.

2. Recombinant DNA Construct With Tissue-Specific Promoter

In this example, a DNA plasmid, referred to herein as pDS GRK1 RHO½L5-14, was produced which has the nucleotide sequence of SEQ ID NO: 6(see FIG. 8 ). This plasmid encodes a RHO ½ meganuclease. The RHO ½meganuclease sequence is operably-linked to a GRK1 promoter, which is atissue-specific promoter that is specifically active in human retinalcells (i.e., rod cells), but not in HEK293 cells used for packaging ofviral vectors. Thus, the RHO ½ meganuclease will not be expressed inviral packaging cells but will be expressed in retinal cells transducedby the virus. Due to the presence of an intron comprising a recognitionsequence for the RHO ½ meganuclease, the viral vector will beself-limiting in the retinal cells as the meganuclease is expressed andcleaves the viral genome.

Example 3 Suppression of Nuclease Expression in Insect Viral PackagingCells Using a Mammalian Intron 1. Rationale

In another example, nuclease expression can be suppressed in insectviral packaging cells by the inclusion of a mammalian intron into thenuclease gene. In this approach, the insect packaging cell cannot splicethe mammalian intron and, consequently, cannot express the nucleaseduring packaging of the viral vector. However, mammalian target cellstransduced with the viral vectors are capable of splicing the intron andexpressing the nuclease, resulting in the degradation of the viralvector in a self-limiting manner.

2, Recombinant DNA Construct

In this example, DNA plasmids were produced which are referred to hereinas pDS CMV RHO ½-HGH (SEQ ID NO: 7; FIG. 9A) and pDS CMV RHO ½-SV40LT(SEQ ID NO: 8; FIG. 9B). Each of these DNA plasmids encodes a RHO ½meganuclease comprising the mammalian human growth hormone intron (SEQID NO: 2), or an SV40 large T intron (SEQ ID NO: 3), respectively.Because insect cells utilize different mRNA splicing motifs thanmammalian cells, these introns are not spliced efficiently from pre-mRNAtranscripts in insect cells. Thus, insect cells will not express afunctional nuclease and will package the full-length genome into a viralvector. Therefore, such DNA plasmids are useful for silencing nucleaseexpression in insect cell expression systems, such as a Baculovirusexpression system, and allowing for the production of self-limitingviral vectors of the invention. In contrast, mammalian cells to whichthe resulting viral vectors are delivered will properly splice thepre-mRNA and will express functional nuclease protein.

Example 4 Promoter Silencing in Viral Packaging Cells Using aRepressor 1. Rationale

Experiments were conducted to further demonstrate that the self-limitingviral vectors of the invention could be successfully generated in viralpackaging cells through the use of a transcription repressor or the useof a tissue-specific promoter. It was hypothesized that a higher titerof intact viral vectors could be achieved due to the suppression ofnuclease activity and a subsequent lack of cleavage at the nucleasebinding site in the viral genome.

2. Transfection of HEK293 Viral Packaging Cells

AAV vectors were produced in HEK293 cells using a standard tripletransfection protocol. Briefly, the helper plasmid pXX680, the pRepCap2plasmid encoding the rep and cap proteins, and a plasmid containing theintended vector genome flanked by AAV2 inverted terminal repeats wereused for cell transfection according to standard protocols. A firstgroup was transformed with the pDS CMV 3xOi RHO ½ L514 LacI plasmid DNAvector (SEQ ID NO: 9), illustrated in FIG. 10A, which comprises anintron containing a RHO ½ binding site, three Lac operators in the CMVpromoter, and a LacI coding sequence for a Lac repressor. A second groupwas transformed with the pDS CMV 3xOi RHO ½ L514 plasmid DNA vector (SEQID NO: 10), illustrated in FIG. 10B, which comprises an introncontaining a RHO ½ binding site, three Lac operators in the CMVpromoter, but lacks a LacI coding sequence. A third group wastransformed with the pDS GRK1 RHO½ L5-14 plasmid DNA vector (SEQ ID NO:6), illustrated in FIG. 8 , which comprises an intron containing a RHO ½binding site and a tissue-specific GRK1 promoter that is specificallyactive in retinal cells (i.e., rod cells). Viral vectors were harvestedfrom cell lysates after 72 hours in culture. Cesium chloride gradientcentrifugation was used to purify AAV vectors from the lysate, whichwere then dialyzed in 1X PBS, aliquoted, and stored at -80° C.

3. AAV Vector Titers

Vector genome copy number (vg) produced in each group was determined bySouthern dot blot analysis using standard protocols. Results aresummarized in Table 1.

TABLE 1 Virus Name Titer Value pDS CMV 3xOi RHO ½ L514 2.40E+07 pDS CMV3xOi RHO ½ L514 LacI 1.10E+08 pDS GRK1 RHO½ L5-14 8.4E+0.7

As shown, the pDS CMV 3xOi RHO ½ L514 vector, which contained nomechanism for suppressing nuclease expression, had a viral titer of2.4×10⁷ viral particles. By contrast, the pDS CMV 3xOi RHO ½ L514 LacIvector, which expressed a Lac repressor to suppress nuclease expressionin the HEK293 packaging cells, had a viral titer of 1.1×10⁸ viralparticles, an increase of approximately 450% compared to the pDS CMV3xOi RHO ½ L5 vector. Further, it was observed that the pDS GRK1 RHO½L5-14 vector having the tissue-specific GRK1 promoter had a viral titerof 8.4x107 viral particles, an increase of approximately 350% comparedto the pDS CMV 3xOi RHO ½ L5 vector.

4. Conclusions

It is evident from these experiments that the inclusion of a mechanismto suppress nuclease expression (e.g., the use of a repressor system, atissue-specific promoter, etc.) allows for a substantial increase in thetiter of intact, self-limiting viral vectors of the invention.

Example 5 Transduction of CHO Cells With Self-Limiting Viral Vectors 1.Rationale

Experiments were conducted using the viral vectors prepared in Example 4to demonstrate that they are self-limiting in a mammalian cell line. Itwas hypothesized that a control vector lacking the nuclease binding sitewould express and accumulate an encoded meganuclease, whereas theself-limiting viral vector would express and accumulate somemeganuclease protein that would ultimately cleave the viral vectors andreduce further nuclease expression.

2. Transduction of Mammalian Cells

CHO cells were either mock transduced, or transduced with the pDS CMV3xOi RHO ½ L514 LacI vector (a self-limiting vector comprising a RHO ½binding site) or the pDS CMV 3xOi RHO ½ L514 vector (not self-limiting;no binding site). AAV vectors were incubated with CHO cells at 100,000viral genomes/cells. Protein was collected at time points of 2, 6, 12,24, 48, and 72 hours using M-Per reagents (Pierce) according to themanufacturer’s instructions. For Western analysis, 50 µg of cell lysatewas resolved on a 4-12% Bis-Tris gel (Invitrogen) according to theprotocol previously described in Example 1 using the anti-I-Crelantibody at a dilution of 1:1000. A separate gel was stained for β-actinas a loading control. Signal detection was assessed bychemiluminescence.

3. Western Analysis

Western analysis is shown in FIG. 11 . The top panel shows detection ofRHO ½ meganuclease protein in cell lysates at each time point. Lane 1shows mock transduced cells. Lanes 2-7 show time points of 2, 6, 12, 24,48, and 72 hours, respectively, for cells transduced with the pDS CMV3xOi RHO ½ L514 vector. Lanes 8-13 show time points of 2, 6, 12, 24, 48,and 72 hours, respectively, for cells transduced with the self-limitingpDS CMV 3xOi RHO ½ L514 LacI vector. The arrow indicates the bandassociated with RHO ½ meganuclease protein. As shown, the RHO ½meganuclease is expressed and continues to accumulate in CHO cellstransduced with the pDS CMV 3xOi RHO ½ L514 vector which is notself-limiting. By contrast, a low level of RHO ½ meganuclease expressionis detectable by 12 hours in CHO cells transduced with the self-limitingpDS CMV 3xOi RHO ½ L514 LacI vector, but this expression level appearsto plateau, presumably because the expressed nuclease recognized andcleaved the binding site in the viral genomes, causing degradation ofthe viral vectors and preventing further expression of the protein.

4. PCR Analysis of Viral Vector Cleavage

To confirm that the self-limiting viral vectors were cleaved intransduced CHO cells, a PCR protocol was developed that utilizes anadapter protein which ligates to the RHO ½ cleavage site of the viralgenome.

Briefly, CHO cells were transduced with the self-limiting pDS CMV 3xOiRHO ½ L514 LacI vector as discussed above and DNA was isolated at 2, 6,12, 24, 48, and 72 hours post-transduction as previously described. Anadapter molecule was designed having a 3′ overhang that matches the 3′overhang generated in the viral genome by the RHO ½ meganuclease. Thus,the adapter would specifically link to the viral genome at a cleaved RHO½ recognition site. 800 ng of DNA was ligated with 2 pmol of adapter at16° C. overnight. PCR was then performed with 200 ng ligated DNA and apair of amplification primers, one matching the AAV sequence, and theother matching the adapter molecule sequence. The resulting PCR productswere analyzed on gel. In case of AAV digestion by RHO ½ and ligation toadapter, a PCR band with size 585 bps was expected to be observed.

5. Results of PCR Analysis

The results of the PCR analysis are shown in FIG. 12 for time points of2, 26, 12, 24, 48, and 72 hours post-transduction. Bands of ~585 bpscorresponding to the correct PCR product are indicated by the arrow. Asshown, band intensity significantly increases from the basal level (2hours) over time, with a maximum signal observed at 24 hours. Bandintensity then appears to be reduced at the 48 and 72 hour time points.These PCR results suggest that the self-limiting viral vectors arecleaved by the expressed RHO ½ meganuclease in transduced CHO cells.

6. Conclusions

These experiments demonstrated that the persistence time of aself-limiting viral vector of the invention, as measured by nucleaseprotein expression, is lower in a transduced mammalian cell line thanthe persistence time of a comparable viral vector that is notself-limiting and does not comprise a nuclease binding site. This issupported by the observation that self-limiting viral vectors arecleaved at the RHO ½ recognition sequence within the viral genome.

What is claimed is:
 1. A viral vector comprising: (a) a first nucleicacid sequence encoding a first engineered nuclease; (b) a first promoteroperably linked to said first nucleic acid sequence, wherein said firstpromoter is positioned 5′ upstream of said first nucleic acid sequenceand drives expression of said first engineered nuclease in a targetcell; and (c) a first vector recognition sequence which is recognizedand cleaved by said first engineered nuclease.
 2. The viral vector ofclaim 1, wherein said viral vector further comprises a first polyAsequence positioned 3′ downstream of said first nucleic acid sequence.3. The viral vector of claim 1 or claim 2, wherein cleavage of saidfirst vector recognition sequence by said first engineered nuclease insaid target cell causes said viral vector to have a lower persistencetime in said target cell when compared to a viral vector which does notcomprise a vector recognition sequence cleaved by said first engineerednuclease but which is otherwise identical.
 4. The viral vector of anyone of claims 1-3, wherein said first vector recognition sequence isidentical to a first chromosomal recognition sequence present in thegenome of said target cell.
 5. The viral vector of any one of claims1-4, wherein said first vector recognition sequence is a sub-optimalrecognition sequence which is recognized and cleaved by said firstengineered nuclease.
 6. The viral vector of any one of claims 1-5,wherein said viral vector further comprises a transgene sequence,wherein said transgene sequence is flanked by sequences homologous tosequences flanking a region of interest in the genome of said targetcell.
 7. The viral vector of claim 6, wherein said transgene sequence ispositioned 5′ upstream of said first promoter.
 8. The viral vector ofclaim 6, wherein said transgene sequence is positioned 3′ downstream ofsaid first nucleic acid sequence.
 9. The viral vector of any one ofclaims 6-8, wherein said first chromosomal recognition sequence ispositioned within said region of interest in the genome of said targetcell.
 10. The viral vector of any one of claims 1-5, wherein said viralvector further comprises a corrected gene sequence, wherein saidcorrected gene sequence does not comprise said first vector recognitionsequence, and wherein said corrected gene sequence corresponds to amutated gene sequence present in the genome of said target cell.
 11. Theviral vector of claim 10, wherein said mutated gene sequence differsfrom said corrected gene sequence by at least one nucleotide andcomprises said first chromosomal recognition sequence.
 12. The viralvector of claim 10 or claim 11, wherein said corrected gene sequence ispositioned 5′ upstream of said first promoter.
 13. The viral vector ofclaim 10 or claim 11, wherein said corrected gene sequence is positioned3′ downstream of said first nucleic acid sequence.
 14. The viral vectorof any one of claims 1-5, wherein said viral vector further comprises asecond nucleic acid sequence encoding a second engineered nuclease. 15.The viral vector of claim 14, wherein said viral vector furthercomprises a second promoter operably linked to said second nucleic acidsequence, wherein said second promoter is positioned 5′ upstream of saidsecond nucleic acid sequence and drives expression of said secondengineered nuclease in said target cell.
 16. The viral vector of claim14 or claim 15, wherein said second nucleic acid sequence is positioned5′ upstream of said first promoter.
 17. The viral vector of claim 14 orclaim 15, wherein said second nucleic acid sequence is positioned 3′downstream of said first nucleic acid sequence.
 18. The viral vector ofany one of claims 14-17, wherein said viral vector further comprises asecond polyA sequence positioned 3′ downstream of said second nucleicacid sequence.
 19. The viral vector of any one of claims 14-18, whereinsaid second engineered nuclease recognizes and cleaves a secondchromosomal recognition sequence present in the genome of said targetcell.
 20. The viral vector of claim 19, wherein said first chromosomalrecognition sequence and said second chromosomal recognition sequenceare positioned on the same chromosome.
 21. The viral vector of claim 19or claim 20, wherein said first chromosomal recognition sequence andsaid second chromosomal recognition sequence flank a region of interestin the genome of said target cell.
 22. The viral vector of claim 19,wherein said first chromosomal recognition sequence and said secondchromosomal recognition sequence are positioned on differentchromosomes.
 23. The viral vector of any one of claims 1-22, whereinsaid first vector recognition sequence is positioned 5′ upstream of saidfirst promoter.
 24. The viral vector of any one of claims 1-22, whereinsaid first vector recognition sequence is positioned 3′ downstream ofsaid first promoter and 5′ upstream of said first nucleic acid sequence.25. The viral vector of any one of claims 1-22, wherein said firstvector recognition sequence is positioned 3′ downstream of said firstnucleic acid sequence.
 26. The viral vector of any one of claims 1-22,wherein said first nucleic acid sequence comprises, from 5′ to 3′, afirst exon, an intron, and a second exon.
 27. The viral vector of claim26, wherein said first vector recognition sequence is positioned withinsaid intron of said first nucleic acid sequence.
 28. The viral vector ofany one of claims 2-22, wherein said first vector recognition sequenceis positioned 3′ downstream of said first nucleic acid sequence and 5′upstream of said first polyA sequence.
 29. The viral vector of any oneof claims 2-22, wherein said first vector recognition sequence ispositioned 3′ downstream of said first polyA sequence.
 30. The viralvector of any one of claims 6-9, wherein said first vector recognitionsequence is positioned 5′ upstream of said transgene sequence.
 31. Theviral vector of any one of claims 6-9, wherein said first vectorrecognition sequence is positioned 3′ downstream of said transgenesequence.
 32. The viral vector of any one of claims 10-13, wherein saidfirst vector recognition sequence is positioned 5′ upstream of saidcorrected gene sequence.
 33. The viral vector of any one of claims10-13, wherein said first vector recognition sequence is positioned 3′downstream of said corrected gene sequence.
 34. The viral vector of anyone of claims 14-22, wherein said first vector recognition sequence ispositioned 5′ upstream of said second nucleic acid sequence.
 35. Theviral vector of any one of claims 15-22, wherein said first vectorrecognition sequence is positioned 3′ downstream of said second promoterand 5′ upstream of said second nucleic acid sequence.
 36. The viralvector of any one of claims 14-22, wherein said first vector recognitionsequence is positioned 3′ downstream of said second nucleic acidsequence.
 37. The viral vector of any one of claims 14-22, wherein saidsecond nucleic acid sequence comprises, from 5′ to 3′, a first exon, anintron, and a second exon.
 38. The viral vector of claim 37, whereinsaid first vector recognition sequence is positioned within said intronof said second nucleic acid sequence.
 39. The viral vector of any one ofclaims 18-22, wherein said first vector recognition sequence ispositioned 3′ downstream of said second nucleic acid sequence and 5′upstream of said second polyA sequence.
 40. The viral vector of any oneof claims 18-22, wherein said first vector recognition sequence ispositioned 3′ downstream of said second polyA sequence.
 41. The viralvector of any one of claims 1-40, wherein said viral vector is anadeno-associated virus (AAV) vector, a retroviral vector, a lentiviralvector, or an adenoviral vector.
 42. The viral vector of any one ofclaims 1-41, wherein said viral vector is an AAV vector comprising a 5′inverted terminal repeat and a 3′ inverted terminal repeat.
 43. Theviral vector of claim 42, wherein said AAV vector is a single-strandedAAV vector or a self-complementary AAV vector.
 44. The viral vector ofany one of claims 1-43, wherein said first promoter is a tissue-specificpromoter, a species-specific promoter, or an inducible promoter.
 45. Theviral vector of any one of claims 1-44, wherein said engineered nucleaseis an engineered meganuclease, a zinc finger nuclease (ZFN), a TALEN, acompact TALEN, or a CRISPR/Cas.
 46. The viral vector of any one ofclaims 1-45, wherein said engineered nuclease is an engineeredmeganuclease.
 47. The viral vector of any one of claims 1-46, whereinsaid first promoter comprises one or more binding sites for atranscription repressor that binds to and silences said first promoter.48. The viral vector of claim 47, wherein said transcription repressoris a Tet repressor, a Lac repressor, a Cre repressor, or a Lambdarepressor.
 49. The viral vector of any one of claims 1-46, wherein saidfirst promoter is an inducible promoter, and wherein said viral vectorfurther comprises a nucleic acid sequence encoding a ligand-inducibletranscription factor which regulates activation of said first promoter.50. A recombinant DNA construct encoding said viral vector of any one ofclaims 1-49.
 51. A recombinant DNA construct encoding said viral vectorof claim 47 or claim 48, wherein said recombinant DNA construct furthercomprises a nucleic acid sequence encoding said transcription repressor.52. The recombinant DNA construct of claim 51, wherein said nucleic acidsequence encoding said transcription repressor is positioned outside ofthe coding sequence of said viral vector.
 53. A method for producing aviral vector, said method comprising transforming a packaging cell withsaid recombinant DNA construct of any one of claims 50-52, wherein saidpackaging cell produces said viral vector.
 54. The method of claim 53,wherein said packaging cell is transformed with said recombinant DNAconstruct of claim 51 or claim
 52. 55. The method of claim 54, whereinsaid recombinant DNA construct further comprises a nucleic acid sequenceencoding said transcription repressor.
 56. The method of claim 55,wherein said nucleic acid sequence encoding said transcription repressoris positioned outside of the coding sequence of said viral vector. 57.The method of claim 54, wherein said packaging cell is furthertransformed with a second recombinant DNA construct comprising a nucleicacid sequence encoding said transcription repressor.
 58. The method ofclaim 54, wherein said packaging cell comprises in its genome a nucleicacid sequence encoding said transcription repressor, and wherein saidpackaging cell stably expresses said transcription repressor.
 59. Themethod of claim 53, wherein said first promoter of said recombinant DNAconstruct is a tissue-specific promoter that is inactive in saidpackaging cell.
 60. The method of claim 53, wherein said first promoterof said recombinant DNA construct is a species-specific promoter that isinactive in said packaging cell.
 61. The method of claim 60, whereinsaid first promoter is a mammalian promoter and said packaging cell is amicrobial cell, an insect cell, or a plant cell.
 62. The method of claim53, wherein said first promoter of said recombinant DNA construct is aninducible-promoter which is regulated by a ligand-inducibletranscription factor, and wherein said recombinant DNA construct furthercomprises a nucleic acid sequence encoding said ligand-inducibletranscription factor.
 63. The method of claim 62, wherein said nucleicacid sequence encoding said ligand-inducible transcription factor ispositioned within the coding sequence of said viral vector.
 64. Themethod of any one of claims 53-63, wherein said packaging cell is aninsect cell, and wherein said first nucleic acid sequence encoding saidfirst engineered nuclease comprises an intron that prevents expressionof said first engineered nuclease in said packaging cell.
 65. The methodof claim 64, wherein said intron is a human growth hormone intron (SEQID NO: 2) or an SV40 large T antigen intron (SEQ ID NO: 3).
 66. Themethod of any one of claims 53-65, wherein said viral vector is an AAVvector, a retroviral vector, a lentiviral vector, or an adenoviralvector.
 67. The method of any one of claims 53-66, wherein said viralvector is an AAV vector.
 68. The method of claim 67, said method furthercomprising transforming said packaging cell with: (a) a secondrecombinant DNA construct comprising a cap gene and a rep gene; and (b)a third recombinant DNA construct comprising adenoviral helpercomponents; wherein said packaging cell produces said AAV vector.